A Multidisciplinary approach to cardiovascular diseases. Proceedings ...

S. NOVO A MULTIDISCIPLINARY APPROACH TO CARDIOVASCULAR DISEASES

SALVATORE NOVO NICOS ANGELIDES - JOHN FLETCHER - KAREL ROZTOCIL

A MULTIDISCIPLINARY APPROACH TO CARDIOVASCULAR DISEASES Proceedings of the st

1 Meeting of the Multinational Chapter of the International Union of Angiology (IUA) in conjunction with

the XXIV Congress of the Mediterranean League of Angiology and Vascular Surgery (MLAVS)

EDIZIONI MINERVA MEDICA

Salvatore Novo Nicos Angelides – John Fletcher – Karel Roztocil

A multidisciplinary approach to cardiovascular diseases Proceedings of the 1 Meeting of the Multinational Chapter of the International Union of Angiology (IUA) in conjunction with the XXIV Congress of the Mediterranean League of Angiology and Vascular Surgery (MLAVS) st

Edizioni Minerva Medica

EDITOR Salvatore Novo Vice-President of the International Union of Angiology for the Multinational Chapter and Vice-President of the Mediterranean League of Angiology and Vascular Surgery CO-EDITORS Nicos Angelides President of the Mediterranean League of Angiology and Vascular Surgery John Fletcher President of the International Union of Angiology Karel Roztocil Past-President of the International Union of Angiology

© 2016 – EDIZIONI MINERVA MEDICA S.p.A. – Corso Bramante 83/85 – 10126 Torino Web site: www.minervamedica.it / e-mail: [email protected]

All rights reserved. No part of this publication may be reproduced, stored in retrieval system, or transmitted in any form or by any means.

Preface

It is a great honor for me to present a selection of papers discussed on the occasion of the 1st Congress organized by the Multinational Chapter of the International Union of Angiology (IUA) in conjunction with the XXIV Congress of the Mediterranean League of Angiology and Vascular Surgery (MLAVS), that was held on October 2-5, 2014 at the Didactic Centre of the Campus of the University of Palermo, with the aim of being an opportunity for scientific dialogue and communication at the highest levels. The subtitle of the meeting “A multidisciplinary approach to cardiovascular diseases” is in agreement with the mission of the IUA and MLAVS, both characterized by a multidisciplinary approach, aiming to provide a unique forum for discussing critical issues concerning the management and care of all vascular patients, that is focused on two main issues: to bring together all types of vascular specialists and support their interdisciplinary cooperation, that is necessary for a good clinical practice at present, and to support young vascular specialists and give them an educational opportunity and the possibility to be scientifically active. During the Congress the most recent experiences and researches in the field of cardiovascular diseases were presented and debated among angiologists, cardiologists, diabetologists, specialists in internal medicine and geriatrics, hematologists, radiologists, vascular surgeons, biologists and genetists, as well as young post-graduate doctors in these disciplines, in a multiform and attractive scientific programme.

Salvatore Novo Chairman of the Congress

Contents

1

35

Ancient medicine and surgery in the Mediterranean N.S. Angelides

The flavone Apigenin counteracts the TNFα-induced endothelial dysfunction and regulates miR-149 expression D. Palmieri, S. Capponi, M. Mura, A. Geroldi, P. Perego, P. Mandich, D. Palombo

6 From peripheral arterial disease to critical limb ischemia in diabetes P. Poredoš

8 Critical limb ischemia: a global controversial problem N.S. Angelides

12 Antiplatelet therapy of patients with peripheral arterial disease P. Poredoš

17 Treatment of distal anastomosis defects during femoro-popliteal below-the-knee bypass with adjunctive intraoperative stenting U.M. Bracale, D. Ferrara, A.M. Giribono, E. Viviani, L. del Guercio, D. Narese, G. Bracale

21 Treatment of femoral pseudoaneurysm after vascular procedure E. Dinoto, F. Pecoraro, D. Pakeliani, G. Bajardi

25 Consensus on intermittent claudication - unsolved problem P. Poredoš, M.K. Ježovnik

27 Secondary prevention of cardiovascular events in pad patients P. Poredosˇ, M.K. Ježovnik

31 How to measure endothelial function? P. Poredosˇ, M.K. Ježovnik

38 Preclinical atherosclerosis and markers of inflammation: do they influence the global cardiovascular risk? S. Novo, V. Bonomo, A. Peritore, P. Carità, G. D’Ambrosio, E. Corrado, G. Novo

41 Molecular markers in coronary artery disease: state of the art C. Bellia, G. Bivona, L. Agnello, B. Lo Sasso, M. Ciaccio

43 Genetic susceptibility to the coronary artery diseases G. Pace, M. Caruso, M. Averna, S. Novo

47 Incretin-based therapies and non alcoholic fatty liver disease A.M. Patti, R.V. Giglio, D. Nikolic, G. Castellino, A. Bonfiglio, V. Mistretta, G. Aurilio, W. Granà, M. Soresi, G. Montalto

50 HCV infection and cardiovascular diseases: understanding the linkage S. Petta, M. Maida, F.S. Macaluso, A. Craxì

53 Correction of cardiovascular risk factors after an acute coronary syndrome: impact of pharmacological therapies on a two-years follow-up N. Manzullo, A. Quagliana, S. Pintacuda, F. Fundarò, D. Piraino, F. Cosentino, S. Novo

56 Impact of anemia on long term prognosis of patients with stemi treated with pci and dual antiplatelet therapy G. Schembri, G. Mazzara, S. Evola, G. Evola, G. Novo, G. Andolina, S. Novo

VI

A multidisciplinary approach to cardiovascular diseases

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Impact of anemia on long-term prognosis of patients with nstemi treated with pci and dual antiplatelet therapy G. Mazzara, V. Evola, G. Schembri, S. Evola, G. Novo, G. Andolina, S. Novo

Baseline analysis on the outcome of patients with deep vein thrombosis (DVT) before the global impact of new oral anticoagulants in Italy: data from RIETE Registry P. Di Micco, A. Visonà , G. Di Micco, A. Guida, D. Jimenez, P. Prandoni, I. Enea, A. Niglio, L. Bertoletti, M. Monreal for the RIETE Investigators

62 Troponin levels after elective percutaneous coronary intervention: outcome and follow-up M. Di Piazza, V. Bonomo, S. Evola, G. Andolina, S. Novo

66 Tdi and myocardial blush: good indicators of myocardial reperfusion M. Meschisi, S. Evola, D. Di Lisi, F. Macaione, A. Peritore, F. Cuttitta, D. Piraino, R. Bonanno, G. Evola, G. Novo, G. Andolina, S. Novo

69 Different strain components, twisting and untwisting: feasibility, reproducibility and pathophysiologic value C. Zito, L. Longobardo, S. Carerj

72 A new clinical classification (V.I.A.) of hypertensive heart disease: our experience P. Candela, E. Bonnì, C. La Greca, C.A. Marrone, D. Buccheri, S. Novo

75 The frequencies of thrombophilic alleles known from GWAS studies in healthy population and in group of patients with venous thromboembolism (VTE) in the Czech Republic J. Kvasnicka, Z. Krska, P. Bobcikova, V. Cverhova, J. Hajkova, I. Malikova, Z. Kudrnova, Z. Zenahlikova, R. Brzezkova, T. Kvasnicka

82 Radiofrequency induced thermal therapy (RFITT): a retrospective analysis A. Haumer, B. Fissneider, S. Haumer

85 Translational lymphology and lymphatic surgery: new advances C. Campisi, M. Ryan, F. Boccardo, C.C. Campisi

88 Prognostic power of atrial indices in stratifying congestive patients P. Barbier

91 Relationship between epicardial fat and diastolic dysfunction in obese subjects: an echocardiographic study G. Novo, M. Meschisi, V. Bonomo, I. Marturana, L. Arvigo, M. Guglielmo, G. Evola, M. Lo Presti, V. Evola, S. Novo, S. Verga

95 Patent foramen ovale (PFO): long-term impact of percutaneous treatment V. Vitale, S. Evola, L. Alioto, R.L. Trovato, G. Novo, G. Evola, G. Andolina, S. Novo

97 State of the art of research on Rivaroxaban: where we are and where we are going G. Vizzari, G. Andò, M. Cusmà Piccione, O. Trio, M.L. Carerj, F. Luzza, G. Oreto, C. Zito


Ancient medicine and surgery in the Mediterranean N.S. Angelides

Founder and Ex-Director of the Department of Cardiac, Vascular and Thoracic Surgery, Nicosia General Hospital, Cyprus Visiting Professor Faculty of Health and Science, Nicosia University, Cyprus President of the Mediterranean League of Angiology and Vascular Surgery

Deep in the past, when ignorance was still the actions of mankind, there was a step by step extraordinary evolution of medicine in the Mediterranean region especially in Greece, Cyprus, Italy and Egypt. This is the reason why I firmly believe that the Mediterranean is the birth place of ancient civilization and in particular of medicine and surgery. Medicine in ancient Egypt-step by step evolution Magic and religion were part of everyday life in ancient Egypt. Gods were considered responsible for many diseases, and because of this any treatment involved, among other procedures, an appeal to a supernatural element, a god, a goddess or a deity. It was impossible at that time to draw a line between the distinct prayers of a priest and the remedies of a healer or physician. Nevertheless, healers were mainly priests and they usually used magic as part of the treatment they offered. The impact of magic is seen in the selection of remedies given to the patients. Ingredients were selected on the basis that they derived from a substance, plant or animal that had characteristics that corresponded in some way to the symptoms of the patient. This principle is known as the principle of simila similibus (“similar with similar”) and is found throughout the history of medicine up to the modern practice of homeopathy! Thus an ostrich egg is included in the treatment of a fractured skull, and a hedgehog against baldness! Amulets were also very popular in ancient Egypt. Health related amulets can be classified in three categories: homeopoetic, prophylactic and theophoric. Homeopoetic amulets demonstrated an animal or part of an animal, from which the wearer hoped to gain strength or speed. Prophylactic amulets were used to protect against harmful gods or demons. Finally, theophoric amulets represented the Egyptian gods who were innumerous. Such a theophoric amulet was considered the girdle of Isis and was intended to stem the flow of blood at miscarriage. Egypt became an organized nation about 3000 B.C. Medicine, as a discipline, appeared later when Egypt had an ambitious Pharaoh named Zoser. Medicine in Egypt was associated with religion and magic. All papyri that had dealt with medicine, gave the “aroma” of

supernatural formulae. But, Medicine in ancient Egypt was not at all primitive! Some medical papyri are really explanatory (Figure 1). In 1822, the translation of the Rosetta stone allowed in parallel the translation of ancient Egyptian hieroglyphic inscriptions. This resulted in an enormous interest towards Egyptology in the 19th century, and led to the discovery of extensive sets of ancient medical documents, including the Ebers papyrus, the Edwin-Smith Papyrus, the Hearst Papyrus, the London Medical Papyrus and others, dating back as far as 2900 B.C. In particular, the Edwin Smith Papyrus is a textbook on surgery and on detailed anatomical observations including the examination, diagnosis, treatment, and prognosis of numerous diseases. It was probably written around 1600 B.C. Medical information in this papyrus dates from as early as 3000 B.C.! Imhotep is credited as the original author of the text and is considered as the founder of ancient Egyptian medicine. In general, in these ancient papyri, many case histories were found. In addition, a great number of internal pathologies were reported, such as eye diseases, diabetes and rheumatism (Figure 2). Later on, physicians in Egypt were organized in hierarchy. They gradually formed a distinct profession, with the court physicians at the top of the hierarchy.

Figure 1. – In the Egyptian writings, Imhotep was presented as a physician and was also considered as the god of medicine.


Figure 2. – Egyptian medical instruments. Figure 4. – Asclepius.

Figure 3. – Homer.

Medicine in ancient Greece The history of ancient Greek medicine begins with “Iliad”. This Homeric poem is considered as the oldest written document in Greek. Ancient Greeks believed that diseases were sent by gods, to punish the mankind. In “Iliad“, Apollo, the god of light, was considered as the disperser of epidemics (Figure 3). Asclepius lived in the thirteenth century B.C. and was considered as the Greek god of Medicine. The symbol of Asclepius was a serpent around a knotted staff. Although Asclepius was a mythological person, solid facts, such as the splendid temples devoted to him, were also present. These places were visited by sick people and that is why the priests of the temples learned the art of healing. The most important “Asclepiion” was situated in the island of Cos, the birth place of Hippocrates, who is considered the father of Medicine (Figure 4). Hippocrates rescued medicine from speculation! He did what no physician had ever done before: he examined the patient, he recorded the signs and symptoms of the disease and gave more emphasis on the evidence than on spiritual causes. He did not ask “Who is causing a particular sickness?” but rather “how and on what process does this particular sickness occur?” He believed that “the best physician is the one who is able to establish a prognosis at the bedside, penetrating the present, the past, and the future of his patient”.

Figure 5. – Hippocrates, the father of medicine.

Hippocrates published about 60 books. Among them is the famous “Oath” every doctor takes at the end of his studies before starting practicing medicine (Figure 5). Medicine after the death of Alexander the Great When the Empire of Alexander the Great was subdivided, the Egyptian share fell to Ptolemy the 1st, who founded Alexandria’s Library under the direction of Aristotle. Ptolemy gathered a number of educated men in Alexandria and organized a system that resembled a University. Anatomy in medicine was inaugurated in Alexandria. Two famous Alexandrian physicians and anatomists were Herophilus (335-280 B.C.), and Erasistratus (280 B.C.). Both took part in the early medical teaching at Alexandria’s University. However, the foundation of Empiricism marked the decline of the Medical School of Alexandria and led to the destruction of an era. Medicine in ancient Cyprus From the “inscription of Idalium” which is the oldest and longest ancient written document found in Cyprus, we learned that Stasikipros, the king of the town of Idalium, signed an agreement with a doctor named Onasilos to treat, free of charge, all soldiers that were


Medicine in ancient Rome

Figure 6. – The inscription of Idalium.

Figure 7. – The famous “warmers”, made from terracotta. These were applied on almost any surface of the body and were filled with fluid. They were very popular as a mean of treatment of various diseases in ancient Cyprus.

wounded in the battles during the siege of Idalium by the Persian army (5th century B.C.). The war ended with an unexpected victory of the Cypriot army and the king honoured Onasilos and his brothers offering them as a price fields and political posts (Figure 6). Another famous Cypriot doctor was Apollonius, who lived in the 1st century B.C. He was well known for his antidotes to various poisons. This is why Mithridates, the king of Pontus, asked him to prepare for him a general antidote, in order to avoid poisoning. This was taken in small doses for a prolonged period and since then this way of treatment was called “mithridadism” (Figure 7).

Greece was the main source of Rome’s knowledge. Roman philosophy and medicine were borrowed from the Greeks, who were well known for their arts and their culture. On the contrary, the Romans were distinguished for their ingenious laws and their governmental status. In the earlier times and before the settlement of Greek doctors in Rome, medical care was under the jurisdiction of the head of the family. The father had great powers by Roman law, and he acted as a physician as well as a judge over his family! The structure of the Roman society in ancient Rome imposed difficulties: the lower classes were poor and the relatively small upper classes were tyrannical, superstitious and cruel. Later on, doctors increased numerically, especially during the reign of Nero. Andromachus was the 1st physician to the Emperor, and received the title of “archiatrus”. In general, there were two classes of archiatri: (a) archiatri sancti palatii, and (b) archiatri populares. The former attended to the Emperor, the latter attended to the people. The most eminent doctor of Rome was Claudius Galen. Galen had probably influenced the progress of medical science with his writings more than any other medical writer. He wrote about 600 books. In his youth he was initiated into the idealism of Plato, the realism of Aristotle and the skepticism of the Epicureans. One of the most important elements in ancient times was water. Its supply was of utmost importance. In ancient Greece, the cities were supplied with water from springs. The Greek aqueducts were simple channels cut in the rock. On the contrary, in Rome the water was carried from great distance with magnificent aqueducts. The important factor about these aqueducts is that no machinery was used to pump the water. In Rome, Thermae and Baths were very important therapeutic places. The baths of Diocletian could for example accommodate 4000 bathers. From a medical point of view, baths in Rome were medically important. They were used for the treatment of various diseases and also for physical culture. However, their legitimate use became gradually limited and their abuse increased and led to the decay of the Roman Empire. Surgery in ancient Egypt In November 2001 in the shadow of the royal pyramid in Sakkara the archeologists made an important discovery. Buried in the sand there was a tomb that had been hidden since 2000 B.C. From the writings found on the walls it became evident that surgery was practiced extensively in Ancient Egypt. However, the first most important evidence about ancient surgery was found in Luxor in 1862. By that time, an archaeologist, named Edwin Smith, used to buy fake papyri, and sell them as genuine. But one of those papyri looked genuine to him. Smith found that this was an ancient medical


treatise and kept it for himself. In 1930’s, this treatise was fully translated and its huge significance was then recognized. The Edwin Smith’s Surgical Papyrus was a detailed writing of surgical treatments for wounds, starting with head injuries. This papyrus described each individual trauma in detail and finally explained how a gaping wound was stitched with the use of acacia thorns as needles and pieces of flax as suturing material! Surgery in ancient Greece Mahaon was considered the first military doctor in Greece. During the war in Troy he offered his medical knowledge for treating wounded heroes. Among his patients was Menelaus, the king of Sparta. However, the wounded heroes most of the times managed to treat their war injuries by themselves (Figure 8). There is a lot of evidence on how surgery was practiced in ancient Greece. Among other operations there is evidence of trepanation: a complex statue in which a surgeon was performing an operation on a patient’s head while god Asclepius, who had a supernatural size, was looking down. It is also of importance that many surgical instruments of the Hippocrates era have great similarities to instruments that are in use today! Surgery during the era of Alexander the Great Alexander was wounded many times, but he always survived and his men began to believe that he was invincible. But invincibility had nothing to do with it. His secret weapon was his surgeon Kritodemos from Cos! During the siege of Multan in Pakistan, Alexander was wounded by an arrow in the chest, which led to extremely inefficient ventilation. The Macedonian leader was placed on a shield and carried to his tent. Unfortunately, he sustained the most lethal kind of injury and the hardest to be treated. But surgeon Kritodemos took action! He decided that the only way to extract the heavy arrow without doing a greater damage was to enlarge the wound. So Kritodemos proceeded with the operation and Alexander went through it without losing

Figure 8. – Achilles is bandaging a war injury for his friend Patroclos during the war in Troy.

his consciousness! After the removal of the arrow, Kritodemos covered and pressed the wound and managed to control the hemorrhage. But by doing this, he also transformed the open pneumothorax to a closed one - a movement that saved Alexander’s life! (Figure 9). Surgery in ancient Rome In ancient times, the demand for surgeons in Rome was great and one of them rose to the top. This was Claudius Galen (130-200 A.D.), who was responsible for many big steps in surgery. On his return to Rome from his voyages abroad, he found that gladiators were valuable and doctors were in great demand to cure their wounds. So Galen decided to practice medicine in Colosseum. There, he was able to see and feel through the gladiators’ wounds, part of the living internal human anatomy, to which other doctors did not have access. Of his 600 books only 20 survived and these were rescued by Arab physicians. Arabs captured and preserved some of the ancient medical texts and from the 9th century they started translating them into Arabic on a massive scale. However, Galen’s revolutionary work did not reach the Western world until the late Middle Ages. But despite of this, many of his theories, as well as a huge number of his remedies were used empirically by doctors for centuries and became the medical equivalent of a holy book!

Figure 9. – Alexander the Great in a fight against the Persians (painting).

Figure 10. – Representation of Galen’s surgery in Rome.


The end of the Great Library of Alexandria and the collapse of the Roman Empire marked the end of the progress in medicine and particularly in surgery for hundreds of years. But, with the coming of the Renaissance, the knowledge of the ancients was rediscovered. Anatomy was accepted, new discoveries in wound treatment were made on the battlefields, and the door to new surgical techniques and practices opened widely. I would like to conclude this short presentation by saying that the fate of medicine and surgery is always the same: an end means the beginning of a new era! References 1. Parkins MD. Pharmacological practices of ancient Egypt. 10th Annual Proceedings of the History of Medicine Days; 2001. 2. Pain S. The Pharaohs’ pharmacists. New Scientist, December 2007, pp. 40-3. 3. Von Staden H. Herophilus: The Art of Medicine in Early Alexandria. Cambridge: Cambridge University Press; 1989. p. 1-26. 4. Temkin O. What Does the Hippocratic Oath Say? In: “On Second Thought” and Other Essays in the History of Medicine. Baltimore: Johns Hopkins University Press; 2002. p. 21-8.

5. Grammaticos PC, Diamantis A. Useful known and unknown views of the father of modern medicine Hippocrates and his teacher Democritus. Hell J Nucl Med 2008;11:2-4. 6. Pećanac M, Janjić Z, Komarcević A et al. Burns treatment in ancient times. Medicinski pregled 2013;66:263-7. 7. Askitopoulou H, Konsolaki E, Ramoutsaki I et al. Surgical cures by sleep induction at the Asclepiion of Epidaurus. The history of anesthesia: proceedings of the Fifth International Symposium, by José Carlos Diz, Avellino Franco, Douglas R. Bacon, J. Rupreht, Julian Alvarez. Elsevier Science B.V., International Congress Series 1242; 2002. p.11-7. 8. Manetti D. Galen’s Library. In: Gill C, Whitmarsh T, Wilkins J (editors). Galen and the World of Knowledge. Cambridge: Cambridge University Press, 2009. 9. Bendick J. Galen and the Gateway to Medicine. San Francisco: Ignatius Press; 2002. 10. Annas J. Classical Greek Philosophy. In: Boardman J, Griffin J, Murray O (editors). The Oxford History of the Classical World. New York: Oxford University Press; 1986. 11. Guthrie WK. A History of Greek Philosophy. Volume I: The earlier Pre-Socratic and the Pythagoreans. New York: Cambridge University Press; 1962. 12. Jones WHS. Philosophy and Medicine in Ancient Greece. Baltimore: Johns Hopkins Press; 1946. 13. Angelides N. Ancient Medicine and Surgery in the Mediterranean. Int Angiol 2014;33(5 Suppl. 1):1.


From peripheral arterial disease to critical limb ischemia in diabetes P. Poredoš

Department of Vascular Disease, University Medical Centre Ljubljana, Ljubljana, Slovenia

Peripheral arterial disease (PAD) is one of the forms of atherosclerotic process for which the major risk factors are similar to those of atherosclerosis in coronary and carotid arteries and other vascular beds. Among traditional risk factors for PAD, diabetes mellitus represents the major risk factor, followed by smoking and advanced age. Diabetes mellitus confirms 1.5-fold to 4-fold increase in the risk in developing symptomatic or asymptomatic PAD and is associated with increased risk for cardiovascular events and early mortality among individuals with PAD.1 In the Framingham Heart Study, 20% of symptomatic patients with PAD were reported to have diabetes. Although, this may have been underestimated because diagnosis was based on reporting of symptoms of intermittent claudication rather than objective testing. 2 In the NHANES (National Health and Nutrition Examination Survey) study, which used the ankle brachial index (ABI) to diagnose PAD, 26% of subjects with PAD were identified as having diabetes.3 While in the Edinburgh Artery Study which used special questionnaire as an objective indicator (ABI less than 0.90), the prevalence of PAD was in individuals with diabetes or glucose intolerance 12.5%.4 The ARIC (Atherosclerosis Risk in Communities) study found that a prior history of diabetes with insulin treatment was independently associated with a greater incidence of PAD. The Multi-Ethnic Study of Atherosclerosis (MESA) found that 26% of women and 23.5% of men with an ABI less than 0.9 have diabetes.5 In patients with diabetes, the prevalence and extent of PAD correlate with the age, duration and severity of diabetes. Diabetes is a stronger risk factor for PAD in women than in man.6 There is 28% increase in the risk of PAD for every percentage point increase in haemoglobin A1c and the seriousness of PAD appears to be related with both: the duration of hyperglycaemia and to glycaemic control.7 PAD prevalence is also increased in individuals with impaired glucose tolerance and the risk of PAD is significantly increased with higher glycated haemoglobin levels. Diabetes is most strongly associated with occlusive disease in the tibial arteries. Diabetes contributes to an increased risk of PAD for different reasons. Persons with diabetes are more likely than non-diabetic patients to have additional risk factors for PAD, such as elevated

blood pressure, tobacco use, increased level of triglycerides, cholesterol and other diseases.4 In addition, diabetes is associated with an increased platelet aggregation, impaired fibrinolytic function and endothelial dysfunction.6 Diabetic patients with PAD are more likely to develop microangiopathy or neuropathy than others or have an increased risk for limb ischemia and limb loss. Critical limb ischemia (CLI) in patients with PAD Clinical presentation of PAD differs and is in up to 50% of patients asymptomatic, particularly in diabetic patients. Other patients develop typical claudication and on average in 1-2% critical limb ischemia. However, it is 3-5 times more frequent in diabetics than in non-diabetics. Critical limb ischemia represents an advanced PAD with serious prognosis, in most of patients within the next six months to a year a major amputation is expected in the absence of hemodynamic improvement (revascularization). The studies showed that the overall prognosis of patients with CLI is serious and in 6 months after diagnosis up to 20% of patients died and only about 45% of Table I. – Factors that increase risk for development of critical limb ischaemia (CLI) and limb loss in patients with peripheral arterial disease (PAD). Factors that increase risk for development of critical limb ischaemia (CLI) and limb loss in patients with peripheral arterial disease (PAD): • f actors that reduce blood flow and oxygen supply of the microvascular bed – diabetes – smoking and tobacco use – anaemia – severe renal failure – severely decreased cardiac output (severe heart failure or shock) – vasospastic diseases or concomitant conditions (e. g. Raynaud’s – phenomenon, prolonged cold exposure) • factors that increase demand for blood flow and oxygen consumption – infection – (e.g. cellulites, osteomyelitis) – skin breakdown or traumatic injury


patients are alive without amputation 8 and 3-years mortality of these patients is higher than 50%. Etiopathogenesis of CLI predominantly depends on the local vascular status, the extent of atherosclerosis and the affection of arteries of lower limbs. In diabetes, typically distal – terminal arteries - are affected which are responsible for the development of the CLI. Beside diabetes, there are other factors which increase the risk for the development of CLI and limb loss in patients with PAD. Particularly smoking and tobacco use, further systemic factors which reduce blood flow and oxygen supply of diseased leg - like anaemia, severely decreased cardiac output, accompanied vasospastic disorders and factors that increase demand for blood flow and oxygen consumption as for example infection (Table I). The other factors which in diabetic patients promote the development of CLI include: accelerated atherosclerotic process because of accompanied risk factors, peripheral neuropathy, injury and inflammation. Because of peripheral neuropathy, injuries with consequent inflammation are more frequent in diabetic than in nondiabetic patients. Further, neuropathy also promotes the development of microangiopathy. How to recognize CLI in diabetic patients? Particularly in diabetes, clinical symptoms and signs of PAD are often unreliable and beside typical pain, weakness of diseased leg is frequently a leading symptom. Further, pathogenesis of ulcers in diabetic patients is very different and they are not only a consequence of deterioration of blood flow, but in some patients, in spite of normal circulation typical neuropathic ulcer develop. The pathogenesis of an ulcer can be recognized by typical clinical appearance (Table II). Differential diagnosis of common foot ulcers is possible in diabetic patients with using simple diagnostic and mostly Table II. – Differential diagnosis of common foot ulcers. Neuropathic ulcer

Neuroischemic ulcer

Painless Normal pulses Typically punches-out appearance Often located on sole or edge of foot or metatarsal head Presence of calluses Loss of sensation, reflexes, and vibration sense Increase in blood flow (arteriovenous shunting) Dilated veins Dry, warm foot Bone deformities Red appearance

Painful Absent pulses Irregular margins Commonly located on toes Calluses absent or infrequent Variable sensory findings Decrease in blood flow Collapsed veins Cold foot No bony deformities Pale, cyanotic

non-invasive tests. These tests include investigation of macrocirculation (ABI, pressure measurement and different imaging tests) and determination of microangiopathy (transcutaneous oxygen measurement (TcPO2) and capillaroscopy). As ABI is non diagnostic, in some diabetic patients because of arterial medial calcination and non-compressibility of peripheral arteries, the toebrachial index is calculated by dividing the toe-pressure by higher of two brachial pressures (TBI). Toe-brachial values below 0.7 are usually considered to be diagnostic for lower extremity PAD and values below 0.30 indicates on the presence of CLI. Useful technique for detection of CLI, particularly in diabetic patients is transcutaneousoxygen measurement. The normal values usually exceed 60 mmHg and if they are below 20 mmHg CLI is expected. Determination of transcutaneous oxygen tension is not important only to identify patients with CLI but has also has a prognostic value and influences therapeutic decision. As imaging techniques are for shank arteries less reproducible and useful, only angiography identifies lesions suitable for revascularization. Conclusion Diabetes represents one of the strongest risk factors for PAD, consequently up to 20% of diabetic patients develop PAD with 2-4 times higher appearance of CLI and amputation rate is seen in diabetic patients, much higher than in non-diabetic. Prevention of CLI includes regulation of blood glucose, management of accompanied risk factors and foot care. Early detection of clinical and preclinical forms of PAD and its aggressive management is of utmost importance. References 1. Kannel WB, McGee DL. Update on some epidemiologic features of intermittent claudication: the Framingham Study. J Am Geriatr Soc 1985;33:13-8. 2. Murabito JM, D’Agostino RB, Silbershatz H et al. Intermittent claudication. A risk profile from The Framingham Heart Study. Circulation 1997;96:44-9. 3. Selvin E, Erlinger TP. Prevalence of and risk factors for peripheral arterial disease in the United States: results from the National Health and Nutrition Examination Survey, 1999-2000. Circulation 2004;110:738-43. 4. MacGregor AS, Price JF, Hau CM et al. Role of systolic blood pressure and plasma triglycerides in diabetic peripheral arterial disease. The Edinburgh Artery Study. Diabetes Care 1999;22: 453-58. 5. McDermott MM, Liu K, Criqui MH et al. Ankle-brachial index and subclinical cardiac and carotid disease: the multi-ethnic study of atherosclerosis. Am J Epidemiol 2005;162:33-41. 6. Association AD. Peripheral arterial disease in people with diabetes. Diabetes Care 2003;26:3333-41. 7. Muntner P, Wildman RP, Reynolds K, Desalvo KB, Chen J, Fonseca V. Relationship between HbA1c level and peripheral arterial disease. Diabetes Care 2005;28:1981-7. 8. Dormandy JA, Rutherford RB. Management of peripheral arterial disease (PAD). TASC Working Group. TransAtlantic InterSociety Consensus (TASC). J Vasc Surg 2000;31:S1-S296.


Critical limb ischemia: a global controversial problem N.S. Angelides

Founder and Ex-Director of the Department of Cardiac, Vascular and Thoracic Surgery Nicosia General Hospital, Cyprus Visiting Professor Faculty of Health and Science Nicosia University, Cyprus President Mediterranean League of Angiology and Vascular Surgery Critical limb ischemia (CLI) is a manifestation of peripheral arterial disease (PAD) that describes patients with chronic ischemic rest pain or patients with ischemic skin lesions, either ulcers or gangrene. The clinical diagnosis of CLI should be confirmed by hemodynamic parameters. The estimated annual incidence of CLI ranges between 500 and 1000 new cases per 1 million, with diabetes being the most important risk factor. CLI is also a marker for mostly generalized and severe atherosclerosis, and therefore the prognosis of patients is poor concerning overall survival. In such cases an urgent revascularization must be attempted without delay. Otherwise, a year after the onset of CLI, 50% of the patients will die mainly as a result of cardiovascular complications, while 40% of the surviving patients will have a major amputation.1 The primary goals of treatment in patients with CLI are to relieve ischemic pain, heal ulcers, prevent limb loss, improve patient function and quality of life and prolong overall survival. Any kind of revascularization should be done whenever technically possible, and therefore most patients should be referred to a vascular center. Furthermore, in patients with CLI a multidisciplinary approach is recommended to control pain, cardiovascular risk factors and other co-morbid disease. In patients with CLI not eligible for arterial revascularization, prostanoids are the only vasoactive drugs with proven efficacy.2 In the last decades, a lot of controversies have appeared in the management of CLI as to whether the newly introduced endovascular treatment should be considered equal or superior to the old open surgical treatment. Several International Multicenter Studies were conducted in an effort to shed light on existing controversial questions. However, the results of these trials have not yet managed to radically solve the problem. CLI: a global controversial problem The everolimus eluting stents vs. bare metal stents (DESTINY) trial 3 showed recently better long-term patency and better limb salvage by using drug eluting stents rather than bare metal stents in patients with CLI and infrapopliteal arterial occlusions. This Study suggested that primary stenting with drug eluding stents is the best method for focal lesions situated below the knee. The lutonix pacli-

taxel coated balloons for the prevention of femoropopliteal restenosis (LEVANT 1 and 2) 4 trials, in an effort to evaluate the safety and efficacy of drug coated balloons in the treatment of femoropopliteal segment occlusion, demonstrated that their use led to a strong inhibition of neointimal hyperplasia. Similarly, the long term results of several other trials supported endovascular treatment in the superficial femoral artery: The randomized comparison of “lifestent” vs. angioplasty (RESILIENT) and the “viabahn” endoprosthesis vs. bare Nitinol stent (VIBRANT) trials (both with more than 3 years follow-up) as well as the recent “heparin-bonded viabahn” stent grafts vs. bare Nitinol stents (VIPER), the short time and exposure for reduction of restenosis in distal arteries (THANDER) trials and the paclitaxel coated balloons in femoral indication to defeat restenosis (PACFIER) trials supply interventional specialists with a lot of information on how to achieve even better and more durable results in the endovascular treatment of the superficial femoral artery.4-8 The use of thrombolysis to overcome acute thrombosis of stenotic infra-inguinal native arteries or of an already performed by-pass graft is an accepted treatment method during the last decade. Compared to standard thrombectomy, catheter directed thrombolysis proved to be less invasive and able to treat small arteries.5 However, there are certain limitations like hemorrhagic complications and distal embolization. The Dutch ultrasound accelerated thrombolysis (DUET trial),9 compared standard catheter-directed thrombolysis (using Urokinase) to ultrasound accelerated thrombolysis. As we know, low intensity ultrasound increases thrombus permeability and fibrin disaggregation. The results are still in progress.10 Concerning vascular trauma in the periphery, it was demonstrated that early management increases the chance of survival of the limb. In most of the cases early fasciotomy was carried out to avoid a compartment syndrome. Recently, patients with peripheral arterial occlusion were treated endovascularly with a vascular robotic system in order to navigate safely and efficiently a flexible catheter system. The results are promising.11, 12 Similarly, the rising era of bio-absorbable stents and their first in-human applications were announced. For this purpose a bio-absorbable vascular device was used to restore blood flow in patients with superficial femoral artery occlusion, releas-


ing at the same time anti-stenotic drugs locally. The bioabsorbable device which dissolves in two years, is made of a bio-compatible material (polytactide) which is used in implants, sutures etc. The question remains whether leaving no metal behind may in the future be the best treatment for superficial femoral artery occlusion.13 Coming back to CLI, tibia angioplasty can be used in most of the cases. Treatment of an isolated below the knee occlusion is predominantly endovascular. However, the presence of multi-level disease should not discourage attempts to percutaneous revascularization. High risk obese patients with minor tissue loss can be treated with balloon angioplasty. However, it is better to immediately operate patients with significant tissue loss, in whom angioplasty failed to create a direct flow, unless major surgical risk exists.14 Finally, in dealing with the coming storm of diabetic vascular disease, it must be stressed that a multidisciplinary approach should be followed for tackling diabetic angiopathy.15 Therapeutic procedures used, include an increasing number of endovascular therapies for revascularization. However, the saphenous vein by-pass remains one of the best options in diabetic critical limb ischemia. It is therefore a fact that the peripheral controversies were and will continue to be watched closely and there is an increasing interest in the below the knee new trends and technologies. In order to classify our own results we carried out the following study. The Cyprus polyvascular Study Our study, the Cyprus Polyvascular Study, is a retrospective multidisciplinary study that involved 696 polyvascular patients (302 females) who had co-existing Metabolic Syndrome. The patients’ mean age was 61±5.3 and all were admitted to the Nicosia General Hospital over a period of 20 years. The following predisposing factors were unusually high: hypertension (87.2%); hyperlipidemia (84.4%); low density cholesterol (LDL) (58.1%). Table 1 shows our results for the aortofemoral and the femoropopliteal segment according to TASC classification: Table I.

Endovascular

Open

TASC A Aorto iliac lesions

96%

4%

TASC B Aorto iliac lesions

92%

7%

TASC C Aorto iliac lesions

60%

40%

TASC D Aorto iliac lesions

15%

85%

TASC A Fem-pop lesions

97%

3%

TASC B Fem-pop lesions

85%

15%

TASC C Fem-Pop lesions

52%

48%

TASC D Fem-Pop lesions

12%

88%

Our results demonstrated that in the presence of type II diabetes, the coincidence of MS and polyvascular arterial disease was characterized by even more severe clinical findings. We also noticed that in the presence of MS there is a significant increase of CLI, either as acute or chronic. We noticed that acute ischemia in CLI was usually the result of embolization or primary thrombosis, which demanded urgent removal of the clot. As a rule, in CLI our indications for open surgery were based on the fact that endovascular attempts failed and there was still a 25% chance of saving a useful limb. By following this principle we avoided repeated unsuccessful attempts, which can put the patient’s life in danger. TASC A, B and C iliac or femoro-popliteal lesions were treated endovascularly. We found that drug-eluting stents showed superiority to bare metal stents in the sense that they have the ability to prevent tissue in-growth for longer periods of time (our re-stenosis rate after a year was less than 10% compared to 34.3% with bare metal stents). Similarly, drug eluting balloons were used efficiently in areas where stents could not be successfully applied; (especially in cases of coronary-in-stent-re-stenosis, superficial femoral artery-in-stent-re-stenosis and in distal blocked arteries). The patency of any graft was checked at the end of the operation. In case of re-occlusion of the graft at a later time we initiated catheter thrombolysis as soon as possible. Successful thrombolysis was followed by correction of the underlying pathology either endovascularly (26% of the cases) or by doing a repeat operation at a second stage (74%). Amputation was decided whenever there was no run off below the femoral artery and there was tissue loss and pain. Such a decision was taken quickly in order to avoid increase of mortality. Rehabilitation was initiated on the day of the amputation. We found that early exercise minimizes oedema and the lack of balance. Prospective Cyprus Polyvascular Pilot Study The results of the retrospective study were discussed at the 4th Lisbon Vascular Forum (DEC 2013). It became apparent that they had the usual weak points that can be spotted in any retrospective study. So, we decided to proceed further to a pilot prospective trial on an isolated group of patients suffering from severe CLI and metabolic syndrome, aiming to compare the results of our old retrospective study to the results of the new prospective study. 71 patients were included in the pilot study. The following differences were noted: (a) the prevalence of hypertension-high cholesterolhigh LDL was significantly lower in the prospective study (Table II). This could be explained by the application of a more aggressive dietary and pharmacological treatment

10


Table II. – Predisposing factors.

(b) Comparison of the endovascular results concerning patency, amputation rate and quality of life between the prospective and the retrospective study showed better values in the prospective study, especially for TASC C and TASC D patients. The one-year-results of endovascular patency are demonstrated on Tables III, IV, V, and VI: Table III. – Iliofemoral (endovascular).

Table IV. – Iliofemoral segment (endovascular treatment).

Table VI. – FEM-POP segment (endovascular treatment).

The one-year amputation rates for the retrospective and prospective study are shown on Table VII. There was a two-fold increase of the amputation rate in patients with CLI and metabolic syndrome who belonged to the retrospective study, as well as a three-fold increase of the same parameter in patients of the retrospective study with CLI, metabolic syndrome and diabetes mellitus (Table VII). Table VII. – One-year amputation rate.

The one-year quality of life was generally improved (Table VIII). However, this improvement was more significant in patients of the prospective than the retrospective study. Also, patients with CLI, metabolic syndrome and diabetes mellitus showed less obvious improvement of their quality of life in comparison to patients with CLI and metabolic syndrome alone (Table VIII). Table VIII. – Improvement one-year quality of life.

Table V. – FEM-POP (endovascular).

(c) Also, it became apparent, that a greater number of patients in the pilot study were having regular supervised exercise that assisted their improvement, to which they cope well. An exception to this rule was a group of insulin dependent diabetic patients


who coped badly with exercise. In our opinion this inability was due to the existing severe diabetic neuropathy and muscular atrophy. (d) Finally, we noticed, the learning curve of the staff performing the endovascular procedures was found significantly higher in the prospective than the retrospective study; on the other hand, the instrumentation used in the prospective study was of a better quality. These findings may explain the improved results found in the prospective study. Our overall results showed that what is really indicated in patients with critical limb ischemia and co-existing metabolic syndrome or diabetes mellitus is an early diagnosis and a proper endovascular treatment with regular follow up. Surgery has always a role to play, especially whenever endovascular treatment has failed or is not recommended. In our opinion, the saphenous vein by-pass alone or in combination with endovascular techniques, still remains the best option in diabetic patients with CLI. References 1. Derubertir BG, Pierse M, Ryer EJ et al. Reduced primary patency rate in diabetic patients after percutaneous intervention: results from more frequent presentation in patients with threatening ischemia. J Vasc Surg 2008;47:101-8. 2. Back MR, Leo F, Cuthbertson D et al. Long-term survival after vascular surgery: specific influence of cardiac function and implications for preoperative evaluation. J Vasc Surg 2004;40:752-68. 3. Bosiers M, Scheinert D, Peeters P et al. Randomized comparison of everolimus-eluting versus bare-metal stents in patients with critical limb ischemia and infrapopliteal arterial occlusive disease J Vasc Surg 2012;55:398-9.

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4. Scheinert D, Duda S, Zeller T et al. The LEVANT I (Lutonix Paclitaxel-Coated Balloon for the Prevention of femoropopliteal occlusion. J Am Coll Cardiol intv 2014;7:10-9. 5. Saxon RR, Chervu A, Jones PA et al. Heparin-bonded, expanded polytetra-fluoroethylene-lined stent graft in the treatment of femoropopliteal artery disease: 1-year results of the VIPER (Viabahn Endoprosthesis with Heparin Bioactive Surface in the Treatment of Superficial Femoral Artery Obstructive Disease) trial. J Vasc Interv Radiol 2013;24:165-73. 6. Diehm N, Pattynama PM, Jaff MR et al. Clinical endpoints in peripheral endovascular revascularization trials: a case for standardized definitions. Eur J Vasc Endovasc Surg 2008;36:409-19. 7. Norgen L, Hiatt WR, Dormandy R et al. Inter-Society consensus for the management of peripheral arterial disease. (TASC II). J Vasc Surg 2007;45 (Suppl. S):S5-67. 8. Bosiers M, Torsello G, Gissler HM et al. Nitinol stent implantation in long superficial femoral artery lesions (DURABILITY I Study) J Endovasc Ther 2009;16:261-9. 9. Tsivgoulis G, Eggers J, Ribo M et al. Safety and efficacy of ultrasound enhanced thrombolysis a review and meta-analysis of randomized and nonrandomized studies. Stroke 2010;41:280-7. 10. Schrijver AM, Reijnen MM, van Oostaven JA et al. Dutch randomized trial comparing standard catheter-directed thrombolysis versus ultrasound-accelerated thrombolysis for thromboembolic infrainguinal disease (DUET): design and rationale. Trials 2011Jan 23;12-20. 11. Bismuth J, Kashef E, Cheshire N et al. Feasibility and Safety of Remote Endovascular Catheter Navigation in a Porcine Model. J Endovasc Ther 2011;18:243-9. 12. Riga CV, Cheshire NJW, Hamady MS et al. The role of robotic endovascular catheters in fenestrated stent grafting. J Vasc Surg 2010:51;810-20. 13. Erne P, Schier M, Resink TJ. The road to bio absorbable stents: Reaching clinical reality? Cardiovasc Intervent Radiol 2006;29: 11-6. 14. White ChJ, Gray WA. Endovascular therapies for peripheral arterial disease Circulation 2007;116:2203-15. 15. Marx N, Grant PJ. Endothelial dysfunction and cardiovascular disease – the lull before the storm. Research 2007;4:82-3.

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Antiplatelet therapy of patients with peripheral arterial disease P. Poredoš

Department of Vascular Diseases, University Medical Centre Ljubljana, Ljubljana, Slovenia

Peripheral arterial disease (PAD) is one of the most prevalent manifestations of atherosclerosis.1 Because of the association with atherosclerosis in other vascular beds, PAD is accompanied with increased morbidity and mortality from myocardial infarction and stroke.2, 3 Therefore, management of patients with PAD should be oriented not only toward solution of local problems related to occlusions of the peripheral vessels, but in prevention of systemic cardiovascular events. Prevention and treatment of atherosclerotic disease in different arterial beds is based on the same or similar measures, including drugs, particularly antiplatelets and antilipemic drugs and ACE inhibitors. The efficacy of these drugs has been investigated in numerous relevant clinical studies and it was shown that using drugs it is possible to significantly reduce cardiovascular events and perioperative mortality.4 particularly in high risk subjects. However, in contrast to coronary artery disease, there have not been any trials including only patients with PAD with sufficient power to estimate precisely the preventive effects of these drugs.5 The role of antiplatelets in management of PAD As with other manifestations of atherosclerosis, platelets and clotting factors play a pivotal role in the progression of PAD and the genesis of cardiovascular complications. Many studies have suggested that patients with PAD manifest platelet hyperaggregability, increased levels of soluble platelet activation markers, enhanced thrombin generation and an altered fibrinolytic potential. Many of the markers characterising the prothrombotic state of PAD are also predictive of future cardiovascular events.6, 7, 8 These data underline the importance of antithrombotic prophylaxis in patients with PAD. Therefore, antiplatelet and antithrombotic drugs represent one of the basic options for prevention and treatment of PAD patients. Aspirin, as the oldest and most frequently investigated antiplatelet drug, in spite of the lack of evidence, was accepted as the basic option also for the management of patients with PAD. The efficacy of aspirin in prevention of cardiovascular events in PAD patients Symptomatic patients The effect of antiplatelet therapy on cardiovascular events has been investigated in numerous studies and it

was first systematically reviewed by the Antithrombotic Trialists’ Collaboration (ATC). A meta-analysis comprising 287 studies compared the efficacy of antiplatelet therapy in approximately 135,000 high-risk patients with vascular diseases, including lower extremity PAD. This report included 42 randomised trials of antiplatelet treatment which comprised 9214 patients with PAD. Among those patients with PAD treated with antiplatelet therapy, there was a 23% relative risk reduction (P90%)1, however it is non-diagnostic in some groups of patients (diabetics), because of calcification and non-compressibility of peripheral the arteries.2 However, recent studies showed that high ABI (>1.4) represents similar risk for cardiovascular morbidity and mortality as low ABI. In the study of Resnick and co-workers.3 which includes 4393 participants with low and high ABI in 8.3-years follow-up, shows that adjusted risk for all-cause mortality was in the group of low ABI (1.69) and in patients with high ABI risk was the same (1.77). How to overcome non-diagnostic value of ABI for determination of PAD? In subjects with typical history, particularly in the presentation of intermittent claudication and high ABI (>1.4), additional investigations should be performed: measurement of toe-pressure,4 imaging techniques, measurement of transcutaneous oxygen pressure (TcPO2)5 and angiography. Measurement of toe pressure is a simple and useful technique. In contrast to shank-arteries where most frequently medio-calcinosis appears, toe-arteries are practically never calcified. Therefore, toe pressure measurement provides more reliable information in this group of patients; however, it has to be taken into account, that values of toe-pressure

are 20-30 mmHg lower than ankle-pressure. Transcutaneous oxygen tender is useful for detection of critical limb ischemia. It is important technique because TcPO2 measures oxygen supply which depends not only on the status of macrocirculation but predominantly microcirculation which is responsible for survival of tissue. In consensus document on management of PAD also imaging diagnostic procedures are not described in details, particularly in the group of patients with atypical clinical presentation. Duplex sonography is useful for detection of PAD, location of atherosclerotic lesions, and determination of the extent of vascular lesions. Beside morphologic information it enables estimation of hemodynamic deterioration which is the consequence of stenosis or occlusion of macro-vessels. However, it is time-consuming technique and investigator-dependent, therefore its reproducibility is limited. Duplex sonography is also less reliable in detection of more distal lesions and in subjects with specific anatomic characteristics. Arteriography (classical, CTA, MRA) represents golden standard for detection of obstructive lesions of complete arterial tree. Using arteriography it is possible to detect atherosclerotic lesions, their exact location and extend of atherosclerotic lesions. The most important indication for the use of arteriography is identification of arterial lesions which are suitable for recanalization procedures. Therefore it is indicated in subjects which are candidates for reopening procedures. But it is an invasive technique with valuable rate of complication. The studies showed that severe complications appear in 0.7% and mortality risk accounts 0.16% (TASC).6 It also offers only morphological information without data on hemodynamic consequences. Further, in some patients with acute and sub-acute occlusions arteries distally from arterial occlusion are not shown in spite of their patency. Weakness of consensus document for secondary prevention of cardiovascular events in PAD patients Aspirin remains the basic drug for management of different atherosclerotic diseases, including PAD. However, recent data indicate that aspirin alone or in combination with dipyridamole does not have a significant

26


effect on reduction of cardiovascular events in PAD patients.7 Meta-analysis from Berger et al.8 which included 5.269 PAD patients did not show significant reduction of risk for myocardial infarction, stroke or cardiovascular death. Aspirin significantly reduces only the risk of secondary endpoint - nonfatal stroke. The reasons for aspirin non-responsiveness in PAD patients are probably different: PAD patients are at highest risk because of the increased atherosclerotic burden and represent advanced form of atherosclerosis. Higher platelet activation in PAD patients in comparison to other atherosclerotic disease was shown and probably it can be the consequence of pharmacogenetic variant in PAD patients. It is expected that new antiplatelet drugs: ticagrelor, picotamide, prasugrel will be more effective than the old antiplatelet drugs. But there are no data on their efficacy in PAD patients.7 Consensus document probably should be also upgraded with recommendation in treatment of PAD with angiotensin converting enzyme inhibitors (ACE). Namely, some recent data indicate that ACE inhibitors not only prevent cardiovascular events but may have an effect on pain free walking distance and quality of life.9 Conclusion Clinical guidelines represent very important and useful tool for providing the efficient, rational and safe medical care. However, they do no cover full variants and treatment options of “non-classical” forms of the disease. PAD represents one of the most frequent manifestations of atherosclerosis, therefore, guidelines should be promptly upgraded with new findings and clinical

experiences. Diagnostic algorithm should be adapted to various types of patients. Each patient should be treated individually, and Consensus document should serve as a general guideline for the best clinical practice for the whole group of patients with proven disease. References 1. McDermott MM, Criqui MH, Liu K et al. Lower ankle/brachial index, as calculated by averaging the dorsalis pedis and posterior tibial arterial pressures, and association with leg functioning in peripheral arterial disease. J Vasc Surg 2000;32:1164-71. 2. Aboyans V, Ho E, Denenberg JO et al. The association between elevated ankle systolic pressures and peripheral occlusive arterial disease in diabetic and nondiabetic subjects. J Vasc Surg 2008;48:1197-203. 3. Resnick HE, Lindsay RS, McDermott MM et al. Relationship of high and low ankle brachial index to all-cause and cardiovascular disease mortality: the Strong Heart Study. Circulation 2004;109:733-9. 4. Park SC, Choi CY, Ha YI et al. Utility of Toe-brachial Index for diagnosis of peripheral artery disease. Arch Plast Surg 2012;39:227-31. 5. Byrne P, Provan JL, Ameli FM et al. The use of transcutaneous oxygen tension measurements in the diagnosis of peripheral vascular insufficiency. Ann Surg 1984;200:159-65. 6. Norgren L, Hiatt WR, Dormandy JA et al. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). J Vasc Surg 2007;45 Suppl S:S5-67. 7. Poredos P, Jezovnik MK. Is aspirin still the drug of choice for management of patients with peripheral arterial disease? Vasa 2013;42:88-95. 8. Berger JS, Krantz MJ, Kittelson JM et al. Aspirin for the prevention of cardiovascular events in patients with peripheral artery disease: a meta-analysis of randomized trials. JAMA 2009;301: 1909-19. 9. Poredos P. Ramipril for treatment of intermittent claudication. Vasa 2013;42:237-8.


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Secondary prevention of cardiovascular events in pad patients P. Poredoš, M.K. Ježovnik


Peripheral arterial disease (PAD) of the lower extremities, is a common form of atherosclerotic disease that is associated with atherosclerosis in the coronary and carotid arteries.1, 2 Although the prognosis of PAD is relatively benign regarding viability of the affected limb, except in diabetics.3 all patients with PAD are at increased risk of myocardial infarction, ischaemic stroke and cardiovascular death.1, 2 Several studies described two-to three fold greater mortality in patients with PAD than in age-matched controls with normal ABI, with a 5-year mortality of about 30%.4 Increasing severity of PAD, expressed as a diminishing ABI progressively reduces survival.5 While great emphasis has been placed on aggressive pharmacological management of coronary artery disease, less attention has been paid to pharmacological management of PAD and for decades PAD patients were undertreated. Although avoiding tobacco and exercising are very valuable, they are by no means enough. Pharmacological interventions in large clinical trials that have included patients with PAD have reduced cardiovascular morbidity and mortality in those subjects at least to a similar degree as in subjects with atherosclerotic disease of other vascular areas.6, 7 This is not surprising since the pathogenesis of atherosclerotic lesions in the peripheral arteries is similar or identical to that in other parts of the circulatory system. In addition to nonpharmacological risk factor elimination, designated pharmacological interventions - similarly as in coronary and cerebrovascular disease - include administration of platelet inhibitors and treatment of hypercholesterolaemia, hypertension and diabetes. Antiplatelet treatment Although there are no trials including only patients with PAD that would have sufficient power to precisely estimate the preventive effect of aspirin, the Antiplatelet Trialists’ meta-analysis confirmed that antiplatelet treatment provides similar systemic benefit in patients with PAD as in other high risk groups, i.e. a reduction of major atherothrombotic events by about one quarter.8 Systemic benefits do not differ between patients with intermittent claudication and patients who have already undergone peripheral revascularization by angioplasty

or bypass surgery. Low dose aspirin, i.e. ≤325 mg/day is as effective as higher doses and are related to decreased risk of gastrointestinal bleeding. However, the last findings and meta-analysis showed that in PAD patients aspirin is less effective than in coronary artery disease. In the subgroup of patients with PAD in the CAPRIE trial, clopidogrel (75 mg/day) was found to be 23,8% more effective than aspirin (325 mg/day) in preventing atherothrombotic events and resulted in less gastrointestinal bleeding.9 Although clopidogrel is generally recognized as slightly more effective as aspirin in preventing major atherothrombotic events in high risk patients, the magnitude of this benefit is statistically uncertain and, mainly due to its much higher cost, clopidogrel has not been accepted as superior by regulatory authorities. Aspirin thus remains the first-line antiplatelet drug and clopidogrel as its effective alternative. Non-responsiveness to antiplatelet therapy Despite antiplatelet treatment, a substantial number of patients continue to suffer atherothrombotic complications. Due to the multifactorial nature of atherothrombosis, no single intervention can abolish all complications, but aspirin and clopidogrel non-responsiveness are emerging clinical entities with increased risk of myocardial infarction, stroke and death. The correct treatment of these conditions remains unknown.10 The CHARISMA trial did not show a benefit of combined aspirin and clopidogrel treatment versus aspirin alone in a combined cohort of subjects with established vascular disease and subjects at high risk, although dual antiplatelet therapy was marginally better in the subgroup with already established vascular disease.11 Lipid lowering Different studies showed that hyperlipoproteinaemia is also a relevant risk factor for PAD. Hypercholesterolaemia was found in 45-59% of symptomatic PAD patients.12 A significant benefit of statin treatment on cardiovascular and cerebrovascular events has been shown in patients with PAD.13 A meta-analysis of randomized lipid-lowering trials in the pre-statin era that included 698 patients with PAD who were treated with a variety of therapies, including diet,

28


cholestyramine, probucol and nicotinic acid for 4 months to 3 years showed that the total mortality was 0·7% in the treated patients, as compared with 2,9% in the patients given placebo - a non-significant difference.14 In the Heart Protection Study (HPS), PAD patients treated with simvastatin had a significant reduction in major vascular events such as myocardial infarction, stroke and cardiovascular death and needed fewer revascularizations.7 In the subgroup of patients with PAD only, the 24·5% proportional reduction in major vascular events with simvastatin in comparison to placebo was very similar to the overall result of the whole trial, a 22% reduction in the event rate. Five years of statin treatment prevented 70 major cardiovascular events in 1000 patients with PAD. This very large randomized treatment trial supports the use of statin drugs in patients without coronary artery disease but with arterial disease of other areas.15 In another, relatively small retrospective study, the use of statins substantially reduced vascular events in PAD patients.13 Fibrates may also be beneficial in PAD. Statins may also have some positive effects on PAD it self. In a subgroup of patients treated with simvastatin in the Scandinavian Simvastatin Survival Study, the relative risk of new claudication or worsening of pre-existing claudication was 0,6 (95% confidence interval, 0·40,9), as compared with patients randomly assigned to placebo (relative risk reduction of 38%).15 All in all, the association between the progression of PAD and lipid abnormalities deserves further study. Well-designed, long-term studies assessing primary and secondary prevention of PAD with defined endpoints, such as amputation rate, number of vascular interventions, or estimates of quality of life are still missing. Haemodynamic effects of statins Some studies also indicated that besides their lipolytic effect statins may directly influence peripheral haemodynamics and improve functional outcomes of PAD patients.16 Lipid-lowering therapy with statins improved the 6-min walk performance, walking velocity and summary performance score in 392 men and women with an ABI < 0,90. In another study, atorvastatin improved the pain-free walking distance and community-based physical activity in patients with intermittent claudication. Therefore, patients with PAD who are treated with statins may experience improvement in walking performance to complement the reduction in cardiovascular events. Managing hypertension Hypertension is one of the most important risk factors for atherosclerotic disease, including PAD. In patients with preclinical and clinical stages of PAD, which is present in 20% of the older population, hypertension

was found in 50-92%.17 In the PARTNERS study, hypertension was more common among patients with PAD or cardiovascular disease than among control subjects without clinically recognized atherosclerosis.18 Follow-up data from the Framingham Study found a 2·54-fold increased risk of PAD in men and women with hypertension.19 Nevertheless, not all studies demonstrated a clear relation between hypertension and PAD. The presence of hypertension additionally increases the risk of cardiovascular events in PAD patients. In a large study from Italy the presence of both hypertension and PAD resulted in an odds ratio of 1·48 for subsequent cardiovascular events. This observation was also confirmed in the SHEP (systolic hypertension in the elderly programme) study where a low ankle-brachial index or = 125 mg/dL treated with statins versus no lipid-lowering drug. Am J Cardiol 2002;90:789-91. 14. Leng GC, Price JF, Jepson RG. Lipid-lowering for lower limb atherosclerosis. Cochrane Database Syst Rev 2000;2:CD000123. 15. Pedersen TR, Kjekshus J, Pyorala K et al. Effect of simvastatin on ischemic signs and symptoms in the Scandinavian Simvastatin Survival Study (4S). Am J Cardiol 1998;81:333-5. 16. McDermott MM, Guralnik JM, Greenland P et al. Statin use and leg functioning in patients with and without lower-extremity peripheral arterial disease. Circulation 2003;107:757-61. 17. Makin A, Lip GY, Silverman S et al. Peripheral vascular disease and hypertension - a forgotten association? J Hum Hypertens 2002;15:447-54. 18. Hirsch AT, Criqui MH, Treat-Jacobson D, et al. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA 2001;286:1317-24. 19. Kannel WB, McGee DL. Update on some epidemiologic features of intermittent claudication: the Framingham Study. J Am Geriatr Soc 1985;33:13-8. 20. Mehler PS, Coll JR, Estacio R et al. Intensive blood pressure control reduces the risk of cardiovascular events in patients with peripheral arterial disease and type 2 diabetes. Circulation 2003;107:753-6. 21. Radack K, Deck C. Beta-adrenergic blocker therapy does not worsen intermittent claudication in subjects with peripheral arte-

rial disease. A meta-analysis of randomized controlled trials. Arch Intern Med 1991;151:1769-76. 22. Warner GT, Perry CM. Ramipril: a review of its use in the prevention of cardiovascular outcomes. Drugs 2002;62:1381-405. 23. Ostergren J, Sleight P, Dagenais G et al. HOPE study investigators. Impact of ramipril in patients with evidence of clinical or subclinical peripheral arterial disease. Eur Heart J 2004;25:17-24. 24. Fox KM, EURopean trial On reduction of cardiac events with Perindopril in stable coronary Artery disease Investigators. Efficacy of perindopril in reduction of cardiovascular events among patients with stable coronary artery disease: randomised, doubleblind, placebo-controlled, multicentre trial (the EUROPA study). Lancet 2003;362:782-8. 26. The Diabetes Contriol and Complications Trial (DCCT) Research Group. Effect of intensive diabetes management on macrovascular events and risk factors in the Diabetes Control and Complications Trial. Am J Cardiol 1995;75:894-903. 27. UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 1998;352:837-53. 28. Selvin E, Wattanakit K, Steffes MW et al. HbA1c and peripheral arterial disease in diabetes. The Atherosclerosis Risk in Communities Study. Diabetes Care 2006;29:877-82. 29. Maca T, Mlekusch W, Doweik L et al. Influence and interaction of diabetes and lipoprotein (a) serum levels on mortality of patients with peripheral artery disease. Eur J Clin Invest 2006;37:180-6.


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How to measure endothelial function? P. Poredoš, M.K. Ježovnik


The endothelium has been recognized as the key regulator of vascular homeostasis. Healthy endothelium produces a wide range of factors that regulate vascular tone, adhesion of circulating blood cells to the vessel wall, thrombus formation, smooth muscle cells proliferation, and vessel wall inflammation which is the key mechanism of the atherosclerotic process. One of the most important functions of endothelium is its effect on vascular tone. This is achieved by production of several vasoactive substances that relax or constrict the vessel, as well as by its response to circulating vasoactive mediators such a bradykinin and thrombin.1 Nitrogen oxide (NO) has the most expressed vasodilating capacity and protective activity; it is synthesized from L-arginine by the action of endothelial NO synthase (eNOS).2 Nitrogen oxide synthesized in endothelial cells diffuses to the smooth muscle cells of the vessel wall and activates guanilate cyclises, leading to vasodilatation. Shear stress is a key activator of eNOS. Endothelium also synthesizes other vasoactive substances such as cytochrome-derived factors, natriuretic peptide and prostacyclin. However, it appears that these substances have more limited roles in maintaining vasodilator activity than NO.3 Nitrogen oxide also maintains vascular wall homeostasis by inhibition of inflammation, cellular proliferation and thrombosis.4 Endothelial dysfunction and atherosclerosis Alteration in endothelial function is one of the earliest measurable markers of deterioration of the vessel wall in atherogenesis which precedes the development of atherosclerotic changes and is a predictor of cardiovascular events.5, 6 The involvement of endothelial dysfunction in atherogenesis is supported by findings which demonstrated that subjects with different risk factors of atherosclerosis such as hypercholesterolemia, diabetes, hypertension and smoking have significantly deteriorated endothelial function.7 The progression of endothelial dysfunction is related to the intensity and duration of proven risk factors, and to the total risk of individual subjects.8, 9 Endothelial dysfunction also plays an important role in the development and growth of atherosclerotic lesions, as well as in the appearance of ischaemia and thrombosis in the late stages of the disease.10

Assessment of arterial endothelial function Endothelial-dependent vasomotion has been the most widely used clinical end-point for assessment of endothelial function. Testing involves pharmacological and/ or physiological stimulation of the endothelial release of NO and other vasoactive substances. Flow-mediated dilation (FMD) Recently non - invasive methods for assessment of endothelial function were introduced. Exploration of the brachial artery flow mediated dilation capability (FMD) is the most commonly used technique for investigation of endothelial function. Flow mediated dilation serves as an index of nitric oxide mediated endothelium-dependent vasodilator function. Endothelial dysfunction is reflected in an unimpaired FMD response.11 As endothelial dysfunction is a systemic process, and correlation between responses in the coronary circulation and in the forearm has been demonstrated,17 testing of the functional-endothelium dependent capability of peripheral arteries represents a window to investigation of the functional status of the whole circulatory system. The capability of endothelium-dependent (flow-mediated) vasodilation (a measure of endothelial dysfunction) is expressed as the change in the final diastolic diameter of the brachial artery during reactive hyperaemia compared with its baseline value. A lack of vasodilation would suggest decreased release of endogenous vasodilators and therefore, endothelial dysfunction. Different protocols have been introduced for measurement of the velocity and diameter of the artery under investigation. The standard and most frequently used technique is determination of the diameter of the investigated artery at a fixed time point (usually 60 seconds after cuff deflation). However, the use of a fixed time frame for FMD measurement has been recently discussed and questioned. Studies demonstrated that the arterial diameter observed at 60s underestimated the maximal FMD response;12 therefore, continuous EKGgated measurement of FMD starting with registration of the diameter before occlusion and then continuing during cuff inflation and for another 3-5 minutes after cuff deflation were suggested.

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Flow-mediated dilation of the brachial artery and the popliteal artery decrease with age, which may be attributable in part to decreased nitrogen oxide release and to diminished smooth muscle cell responsiveness in older subjects.13 Since flow-mediated vasodilation is expressed as the change in post-stimulus diameter as a percentage of the baseline diameter, baseline diameter influences the change during stimulation. A larger baseline diameter yields a smaller percentage of change, and smaller arteries appear to dilate relatively more than do larger arteries.14 Flow-mediated dilation measurement has also become popular in clinical studies, because it strongly predicts cardiovascular events in patients with established cardiovascular disease. These studies generally indicated that FMD provides independent prognostic information, which may exceed the predictive value of traditional risk factors. In summary, FMD appears to be predictive of cardiovascular events in asymptomatic subjects and in those with established cardiovascular diseases. FMD is not only as predictive as traditional risk factors but may also provide important additional prognostic individual information on the risk of cardiovascular events.13 Therefore, assessment of endothelial function on the basis of FMD has been proposed as a possible non-invasive and inexpensive endpoint that could reflect the cumulative cardiovascular burden and the responsiveness to therapies in individual patients. Low-flow-mediated constriction of conduit arteries Flow-mediated dilation is a measure of vasodilation capability in response to a sudden increase in shear stress, and as such, quantifies the capacity of the endothelium to cause smooth muscle cells relaxation and vasodilation when stimulated by a specific stimulus.16 To investigate vascular reactivity at rest, a new method was developed - low-flow-mediated constriction (L-FMC). Low-flowmediated constriction quantifies the decrease in forearm conduit artery diameter that occurs in response to a decrease in blood flow and shear stress.17 The measurement of L-FMC is usually combined with determination of FMD, and a composite end-point of the absolute value of FMD and L-FMC is calculated. The methods employed for L-FMC/FMD determination differ in investigation of different arterial segments, i.e. for brachial vs. radial arteries. These findings indicated that different mechanisms are responsible for basal and stimulated vasoactive response to shear stress. While the mechanisms underlying FMD are based on synthesis and release of NO, the absence of inhibition of the synthesis of NO does not modify L-FMC, suggesting that NO has a less important role in maintaining the resting tone of arteries. The data shows that in resting conditions other substances such as prostaglandin are secreted by the en-

dothelium of conduit arteries and that this production is decreased when shear stress is reduced. Therefore, L-FMC might be a result of the common effect of vasodilator inhibition (prostaglandins, endothelium-derived hyperpolarizing factor) and increased endothelin-1 production. It was also indicated that simultaneous measurement of FMD and L-FMC provides more comprehensive information on the different pathways involved in control of vascular homeostasis. Peripheral arterial tonometry Peripheral arterial tonometry (PAT) evaluates the finger arterial pulse wave amplitude. PAT is a non-invasive technique that enables the plethysmographic recording of the pulse wave amplitude (PWA) which is a measure of pulsatile volume changes. Pulse wave amplitude is assessed before and during reactive hyperaemia. The baseline PWA is determined using plethysmographic finger cuffs placed simultaneously on the index fingers of both hands, for a period of 5 minutes. Hyperaemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff. And the PWA - reactive hyperaemia index is calculated as the ratio of the average PWA between post- and preocclusion values. These values are normalized to measurements of the contralateral arm, which serves as a control for non-endothelial-dependent systemic effects of reactive hyperaemia.18 The physiological mechanisms underlying PWA response are not completely understood. Although some studies have demonstrated a direct contribution of nitric oxide to both the brachial artery FMD and the digital PAT values, conflicting results regarding a correlation between these two investigative methods have been reported. Although increases in peripheral artery blood-flow after reactive hyperaemia are mostly dependent on nitric oxide bio-availability, changes of blood flow are widely recognized as an index of micro-vascular and not of macro-vascular function. Studies have also demonstrated that a higher basal brachial artery blood flow might be an indicator of a hyperperfusive state and underlying vascular dysfunction, and be associated with risk factors and the presence of cardiovascular disease.19 These findings suggest that the digital PAT ratio and brachial artery FMD assess distinct vascular functions. Abnormalities of the pulse wave amplitude have long been described in the peripheral circulation of patients with atherosclerosis. Furthermore, PAT hyperaemia has been shown to be an adequate surrogate marker to access changes in vascular function over time, and in the Framingham cohort it was closely linked with cardiovascular risk factors. Studies also indicated a relationship between coronary endothelial dysfunction as detected by invasive evaluation of the coronary arteries and the findings of PAT; therefore, PAT potentially of-


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Table I. – Validity and differences between different methods investigating of endothelial function / dysfunction. Technique

Target of investigation

Utility

Arterial system Fmd

large, conduit arteries (peripheral, coronary)

Determination of ED of large arteries and its relationship to RF of atherosclerosis and atherosclerotic disease

L-fmc

Peripheral - conduit arteries

Measurement of endothelial activity at rest - during decreased blood flow and shear stress. Provides information on different pathways involved in regulation of vascular tonus

Pat

Small (digital) arteries and microcirculation

Calculation of microvascular function and detection of hyperperfusione syndrome and underlying vascular dysfunction

Dorsal hand vein technique

Dorsal hand vein and other peripheral veins

Evaluation and quantification of functional responsiveness of peripheral veins

Radionuclide venous pletismography

Venous forearm capacitance veins

Registration of forearm blood flow and assessment of venous tone or venous compliance

Venous system

FMD: flow mediated dilation, L-FMC: low flow - mediated constriction, PAT: peripheral arterial tonometry

fers a more simplified non-invasive measurement; however, it measures vascular functions other than FMD. Applicability and utility of different methods for investigation of endothelial function Endothelial dysfunction is a key underlying factor in the atherosclerotic process and the earliest measurable functional abnormality of the vessel wall in atherogenesis. Therefore, different tests of endothelial function have been sought, particularly those involving disturbed endothelium-dependent vasomotion. All available methods aim to evaluate endothelial function/dysfunction; however, they differ in investigating the mechanisms of endothelial dysfunction. Further, different techniques investigate the functional capability of various sections of the circulatory system (Table I). Only some of these methods have been standardized and accepted for routine clinical use. Endothelial-dependent flow-mediated dilation of conduit arteries was one of the first non-invasive methods for clinical assessment of endothelial function. It is an indicator of the vessel wall dilation capability of large conduit arteries. Studies have confirmed the close interrelationship between FMD and total cardiovascular risk and the extent of atherosclerosis. Flow-mediated dilation process is based on NO release from endothelial cells; therefore, blunted dilation is expected in subjects with increased oxidative stress as the consequence of the presence of risk factors of atherosclerosis. Low-flow-mediated constriction of conduit arteries quantifies the decrease in forearm conduit artery diameter that occurs in response to decreased blood flow

and shear stress. This method is not predominantly based on NO availability but it might be mediated by other substances and is probably a common effect of the release of different vasodilators: prostaglandins and endothelium-derived hyperpolarizing factor. Therefore, L-FMC provides different information than FMD and is used as a complementary technique for measurement of endothelium-dependent flow-mediated dilation. Peripheral arterial tonometry is based on plethysmographic recording of the pulse wave amplitude. The physiological mechanisms underlying PVA response are not understood. Studies indicated that, as in FMD, NO plays an important role in digital PAT. Changes in blood flow registered by PAT are indicators of microvascular but not macrovacular function; therefore, digital PAT assesses vascular function distinct to that investigated by FMD. According to this presumption, PAT would be useful for the determination of endothelial dysfunction at the level of microcirculation and resistance arteries in diseases accompanied by microangiopathy; however, the utility and value of PAT measurement are less established than FMD. References 1. Deanfield JE, Halcox JP, Rabelink TJ. Endothelial function and dysfunction: testing and clinical relevance. Circulation 2007;115:1285-295. 2. Forstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 2006;113:1708-14. 3. Moncada S, Higgs EA, Vane JR. Human arterial and venous tissues generate prostacyclin (prostaglandin x), a potent inhibitor of platelet aggregation. Lancet 1977;1:18-20.

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4. Moncada S, Erusalimsky JD. Does nitric oxide modulate mitochondrial energy generation and apoptosis? Nat Rev Mol Cell Biol 2002;3:214-20. 5. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-9. 6. Poredos P. Endothelial dysfunction in the pathogenesis of atherosclerosis. Int Angiol 2002;21:109-16. 7. Drexler H, Hornig B. Endothelial dysfunction in human disease. J Mol Cell Cardiol 1999;31:51-60. 8. Poredos P, Kek A. Relation of blunted dilation of the brachial artery in insulin-dependent diabetes mellitus to microalbuminuria. Am J Cardiol 2000;86:364-7. 9. Poredos P, Orehek M, Tratnik E. Smoking is associated with dose-related increase of intima-media thickness and endothelial dysfunction. Angiology 1999;50:201-8. 10. Poredos P. Endothelial dysfunction and cardiovascular disease. Pathophysiol Haemost Thromb 2002;32:274-7. 11. Thijssen DH, Black MA, Pyke KE et al. Assessment of flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol 300:H2-12. 12. Black MA, Cable NT, Thijssen DH et al. Importance of measuring the time course of flow-mediated dilatation in humans. Hypertension 2008;51:203-10.

13. Parker BA, Ridout SJ, Proctor DN. Age and flow-mediated dilation: a comparison of dilatory responsiveness in the brachial and popliteal arteries. Am J Physiol Heart Circ Physiol 2006;291:H3043-9. 14. Celermajer DS, Sorensen KE, Gooch VM et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340:1111- 5. 15. Inaba Y, Chen JA, Bergmann SR. Prediction of future cardiovascular outcomes by flow-mediated vasodilatation of brachial artery: a meta-analysis. Int J Cardiovasc Imaging 26:631-40. 16. Corretti MC, Anderson TJ, Benjamin EJ et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002;39:257-65. 17. Gori T, Dragoni S, Lisi M et al. Conduit artery constriction mediated by low flow a novel noninvasive method for the assessment of vascular function. J Am Coll Cardiol 2008;51:1953-8. 18. Heffernan KS, Karas RH, Patvardhan EA et al. Peripheral arterial tonometry for risk stratification in men with coronary artery disease. Clin Cardiol 2010;33:94-8. 19. Allen JD, Wilson JB, Tulley RT et al. Influence of age and normal plasma fibrinogen levels on flow-mediated dilation in healthy adults. Am J Cardiol 2000;86:703-5, A709.


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The flavone Apigenin counteracts the TNFα-induced endothelial dysfunction and regulates miR-149 expression D. Palmieri a,b, S. Capponi c, M. Mura a,b, A. Geroldi c, P. Perego b,d , P. Mandich c and D. Palombo a,b

Vascular and Endovascular Unit, Laboratory of Clinical and Experimental Vascular Biology, University Hospital IRCCS San Martino, Genoa, Italy b Research Center of Biologically Inspired Engineering in Vascular Medicine and Longevity (BELONG), University of Genoa, Genoa, Italy c Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics and Maternal and Child Health-Section of Medical Genetics, University Hospital IRCCS San Martino, Genoa, Italy d Department of Civil, Chemical and Environmental Engineering, University of Genoa, Genoa, Italy a

Many epidemiologic and molecular studies have showed a decreased risk of cardiovascular disease associated with a higher intake of polyphenol and flavonoid compounds.1 Apigenin, a flavone naturally occurring in fruits and vegetables, has strong antioxidant, antiinflammatory and anticancer activity.2 However little is known about its effects on endothelial dysfunction. Using an in vitro model of endothelial cell activation by the pro-inflammatory cytokine TNFα,3 we investigated the effects of Apigenin on the expression of endothelial dysfunction markers, including Metalloproteinase-9 (MMP-9), Interleukin 6 (IL-6) and Interleukin 10 (IL10), and focused on microRNAs, a class of non-coding small RNAs that negatively regulate the expression of proteins at the post-transcriptional level. Materials and methods EAhy926 endothelial cells were treated or not with Apigenin (50 µm) for 1 h and then with TNFα (1 ng/mL). The experiments were performed twice. MMP-9 activity was measured by gel zymography, IL6 and IL-10 by Elisa assay. To evaluate the involvement of microRNAs in the protective effects of Apigenin we performed the in silico

analysis for microRNA target prediction using miRanda software (http://www.microrna.org/microrna/home.do). The expression of the selected microRNA (miR-149) was evaluated by quantitative real time-polymerase chain reaction (RT-qPCR) and its regulatory effect was evaluated by gain-of-function technique upon specific mimics transfection. Statistical analysis was performed using ANOVA and Fisher test. P