Vascular tenascin-C regulates cardiac ... - The FASEB Journal

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The FASEB Journal express article 10.1096/fj.05-5131fje. Published online February 6, 2006.

Vascular tenascin-C regulates cardiac endothelial phenotype and neovascularization Victoria L. T. Ballard,* Arti Sharma,* Inga Duignan,* Jacquelyne M. Holm,* Andrew Chin,* Ruby Choi,* Katherine A. Hajjar,† Shing-Chiu Wong,* and Jay M. Edelberg*,† *Department of Medicine and †Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 520 East 70th Street, New York, New York *Corresponding author: Jay Edelberg, Department of Medicine, Division of Cardiology, Weill Medical College of Cornell University, 520 East 70th Street, New York, NY 10021. E-mail: [email protected] ABSTRACT Microenvironmental cues mediate postnatal neovascularization via modulation of endothelial cell and bone marrow-derived endothelial progenitor cell (EPC) activity. Numerous signals regulate the activity of both of these cell types in response to vascular injury, which suggests that parallel mechanisms regulate angiogenesis in the vascular beds of both the heart and bone marrow. To identify mediators of such shared pathways, in vivo bone marrow/cardiac phage display biopanning was performed and led to the identification of tenascin-C as a candidate protein. Functionally, tenascin-C inhibits cardiac endothelial cell spreading and enhances migration in response to angiogenic growth factors. Analysis of human coronary thrombi revealed tenascin-C protein expression colocalized with the endothelial cell/EPC marker Tie-2 in intrathrombi vascular channels. Immunostains in the rodent heart demonstrated that tenascin-C also colocalizes with EPCs homing to sites of cardiac angiogenic induction. To determine the importance of tenascin-C in cardiac neovascularization, we used an established cardiac transplantation model and showed that unlike wild-type mice, tenascin-C–/– mice fail to vascularize cardiac allografts. This demonstrates for the first time that tenascin-C is essential for postnatal cardiac angiogenic function. Together, our data highlight the role of tenascin-C as a microenvironmental regulator of cardiac endothelial/EPC activity. Key words: heart • bone marrow • angiogenesis • EPC

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ostnatal vascular remodeling in response to tissue injury requires a combination of both systemic and local angiogenic mechanisms, i.e., the recruitment of circulating endothelial progenitor cells (EPCs) and remodeling of the existing vasculature (1). In the vasculature, angiogenesis involves the detachment, proliferation, migration, and assembly of endothelial cells at sites of damage to form a collateral blood supply (2). In the case of myocardial infarction, this new vascular network ramifies through damaged myocardium to form new conduits for blood flow. Specifically, in response to coronary thrombus formation, a temporary vascular plexus forms through the clot itself to allow blood to move through the occluded vessel, until the clot is broken down via fibrinolytic mechanisms (3). At the same time, EPCs are mobilized from the Page 1 of 22 (page number not for citation purposes)

bone marrow into the systemic circulation (4). These cells must then home to the site of injury, where they either attach to the endothelium and incorporate into the newly remodeling vasculature or provide support for local angiogenesis via expression of angiogenic growth factors (5, 6). It is known that a variety of local microenvironmental signals mediate these angiogenic mechanisms, including integrins, proteases, chemokines, and cytokines (7). Many of these factors play important roles both at local sites of vascular remodeling, e.g. after myocardial ischemia, as well as in the bone marrow at the site of EPC mobilization. These factors include stromal-derived factor-1 (8), granulocyte-macrophage colony-stimulating factor (9), and vascular endothelial growth factor (VEGF) (10). Based on the parallels between the bone marrow and heart, we hypothesized that these two vascular beds employ similar microenvironmental pathways to regulate angiogenesis in response to vascular damage. Hence, the identification of factors commonly expressed at the vascular surface in the bone marrow and heart may provide novel targets for the development of therapeutics for the treatment and possible prevention of cardiovascular disease. We have previously demonstrated that cardiac vascular repair mechanisms are dysregulated with age, due to the angiogenic impairment of both cardiac microvascular endothelial cells (CMECs) and bone marrow-derived EPCs (11, 12). In light of this impairment, a functional proteomic approach involving in vivo phage biopanning was undertaken to identify vascular epitopes preferentially expressed in the young animal that could contribute to the regulation of EPC/cardiac endothelial cell function. Here, we report the identification of tenascin-C as a mediator of postnatal cardiac angiogenic mechanisms. MATERIALS AND METHODS Studies employing neonatal, 3- and 18-month-old C57Bl/6 mice, 3-month-old ROSA-26 and tenascin-C–/– mice and 4-month-old F344 rats were performed in compliance with the Institutional Animal Care and Use Committee of Weill Medical College. Studies employing human coronary thrombi were performed in compliance with the Internal Review Board of Weill Medical College. In vivo phage biopanning An in vivo phage biopanning study was performed with the aim of identifying epitopes that may play important roles in regulating endothelial cell and/or EPC phenotype and subsequent cardiac angiogenic mechanisms. A multivalent phage library encoding a cyclically constrained 6 amino acid variable region ~107 total complexity; New England Biolabs) was injected into both 3- and 18-month-old mice (n=4, each), as described previously (13) to identify young-specific bone marrow-homing phage. Mice were anesthetized with 2.5% Avertin and injected with the phage peptide library (1010 colony-forming units/100μl PBS) via the tail veins. After 4 min, mice were killed, and bone marrow was extracted. Phage were recovered with WK6λmutS E. coli. Agespecific phage pools were amplified and titrated for two additional rounds of enrichment. Phage clones sequenced with higher frequency from phage pools eluted from young compared with old bone marrow were injected individually to confirm preferential binding in the young bone marrow. Young bone marrow-homing phage were then analyzed for cardiac binding titers to

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identify phage epitopes preferentially binding to the young cardiac vasculature as well as bone marrow. The number of bound phage recovered per milligram of tissue was quantified by serial dilution titration. The preferential cardiac binding capacity of a candidate phage clone, ψR3Y32, in the young compared with old hearts was confirmed by serial dilution as well as immunohistochemical analysis. Cardiac microvascular endothelial cell isolation and in vitro analyses CMECs were isolated from 4-month-old rats according to the method of Nishida et al. (14). Cells were cultured on either collagen-, tenascin-C-, or fibronectin-coated 8-well chamber slides (Nunc Nalge International; rat collagen type-I and human fibronectin from Sigma, chicken tenascin-C from Chemicon, Temecula, CA; 10 μg/ml substrate concentration) in minimal media (5% fetal bovine serum, 1% penicillin, 1% streptomycin in DMEM) for up to 48 h and photographed daily. All experiments were replicated with independent isolations of cells. For cell adhesion analysis, media was removed from the wells 10 min or 3 h after plating and the number of cells that had failed to attach was counted using a hemocytometer. For migration analysis, cells were covered with 300 μl of collagen type-I gel (Chemicon) after 3 h of culture on collagen or tenascin-C. The gel was allowed to solidify for 1 h at 37°C and 200 μl of minimal media containing VEGF-A or PDGF-AB (5 ng/ml) or no growth factor was added to the wells. Cells were incubated for up to 48 h and were analyzed for peak migration rates using an inverted microscope: migratory distance of the 5 cells that had migrated furthest through the z-axis of the gel was determined using the z-plane focus knob of the microscope. Each experiment was performed four times in duplicate. Intracardiac bone marrow homing Bone marrow cells were isolated from 3-month-old, genetically labeled reverse orientation splice acceptor-26 [ROSA-26; (15)] mice as described previously (12). Approximately 107 bone marrow cells were injected via the tail vein into irradiated 3-month-old C57Bl/6 mice (9 Gy radiation, 3 h before bone marrow injection). After 28 days, cardiac injections of PDGF-AB were performed, as described previously (11). Briefly, mice received intramyocardial injections of PDGF-AB (100 ng/50 μl PBS) at the anterior wall of the myocardium (two 25 μl injections, 2 mm apart) through a 28-gauge needle. After 24 h, mice were killed and the hearts were harvested for paraffin embedding and immunohistochemical analysis. Neonatal cardiac allograft assay A syngeneic cardiac allograft model (11, 12) was used to study fibrin neovascularization and cardiac angiogenesis in wild-type C57Bl/6 and tenascin-C–/– mice [the kind gift of Dr. Moriaki Kusakabe, Jikei University, Japan (16)]. Briefly, unirradiated 3-month-old host mice were anesthetized and a subcutaneous pocket created by blunt dissection into which a heart from a 1day-old C57Bl/6 mouse was directly inserted, as described previously (11, 12). Sets of mice were killed 3 days post-transplantation to examine fibrin clot resolution and vascularization (n≥6 transplants per group). In additional sets of mice, allograft viability was scored 7 days posttransplantation by visual observation and electrocardiogram (ECG) activity, as described previously (11, 12). Mice were then killed for immunohistochemical analysis.


Cardiac transplant studies were also performed in unirradiated 18-month-old C57Bl/6 mice receiving bone marrow cells isolated from 3-month-old syngeneic ROSA-26 and tenascin-C–/– mice. Approximately 107 bone marrow cells, isolated as described above, were injected via the tail vein. After 7 days, a neonatal cardiac transplantation was performed for assessment of cardiac angiogenic potential and histological analysis (n≥12 transplants per group). Immunohistochemistry The following primary antibodies were used: anti-tenascin-C (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pIII (Mobitec, Gottingen, Germany), anti-β-galactosidase (Biogenesis, Poole, UK), and anti-PDGFRα (Santa Cruz). All immunostains were performed on sections from at least three animals. For consistency, heart sections for all analyses were selected from the midpapillary region of the ventricles. For bone marrow immunohistochemistry, femurs were removed, fixed, and decalcified and then processed for paraffin embedding according to standard procedures. Primary antibodies were labeled with biotin (Biogenex, San Ramon, CA) and visualized with Texas Red- and FITC-conjugated avidin (Vector Labs, Burlingame, CA) or labeled with peroxidase and visualized with Vector VIP substrate or diaminobenzidine (DAB) and Giemsa co-staining. For co-staining using two biotinylated primary antibodies, antibody incubations were performed sequentially. Prior to addition of the second biotinylated antibody, a blocking step involving incubation of the sections with three changes of avidin was performed, followed by extensive washing with PBS-Tween 20 to block all exposed biotin sites. RT-PCR expression analysis Rat CMECs were cultured on 0.1% gelatin-coated 12-well plates and incubated with PDGF-AB (50 ng/ml) in minimal media for 0–24 h. Experiments were performed four times from independent cell isolations. RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from ∼2 μg total RNA using Omniscript Reverse Transcriptase (Qiagen) in a 20 μl reaction, according to the manufacturer’s instructions. PCR was performed using 2 μl cDNA, 5 pmol of each primer per 25 μl reaction and 2X Hotstar PCR mastermix (Qiagen). The following cycling program was used: 95°C (5 min) followed by 30 cycles of 94°C, 55°C, 72°C (45 s each), and finally 72°C, 10 min. Relative mRNA levels were compared with corresponding levels of βactin by densitometry and confirmed by quantitative RT-PCR. The primer pairs used were as follows: β-actin: forward, 5′-GTCGTACCACTGGCATTGTG-3′; reverse, 5′-ACCCTCATAGATGGGCACAG-3′; tenascin-C: forward, 5′-GTTTGGAGACCGCAGAGAAGAA-3′; reverse, 5′-TGTCCCCATATCTGCCCATCA-3′ Clinical intracoronary samples Intracoronary thrombi were obtained from percutaneous intracoronary thrombectomy procedures performed during primary angioplasty for acute coronary syndromes in 4 individuals (ages 58–


65). Aspirated intracoronary thromboocclusive material was collected into the thrombectomy devices (Export Catheter, Medtronic and Filterwire, Boston Scientific) and immediately placed in formalin, fixed for 24 h at 4°C, and processed for paraffin embedding. Statistical analysis All studies were performed in triplicate unless otherwise stated. Statistical analysis of the degree of cell affinity to tenascin-C and collagen in vitro was performed using the unpaired Student’s t test. For analysis of in vitro cell migration, a two-way ANOVA with a post-hoc multiple comparison test was performed. Cardiac allograft viability was analyzed using the Chi-squared test. RESULTS Identification of a phage epitope preferentially binding in the young bone marrow and cardiac vasculature An in vivo phage biopan was performed with the aim of identifying epitopes that may be involved in common mechanisms of angiogenesis in the heart and bone marrow. Based on the age-related impairment in both cardiac endothelial and EPC-mediated angiogenic pathways (11, 12, 17), we focused on phage epitopes that preferentially bind at the vascular surface in the young vs. old host. Three rounds of panning resulted in enrichment of a phage clone, designated ψR3Y32, from the bone marrow and heart of young mice. Individual injection of ψR3Y32 confirmed 10- and 6-fold higher levels of ψR3Y32 binding in young compared with old murine hearts and bone marrow, respectively (Fig. 1A). Injection of control, non-recombinant phage demonstrated negligible levels of binding (data not shown). This finding suggested that binding partners for ψR3Y32 are preferentially expressed in the vasculature of the young bone marrow and heart. Comparison of the variable amino acid region of ψR3Y32 to known extracellular peptide sequences revealed homology of this phage epitope to an extracellular domain of α8-integrin. This integrin has a number of known binding partners, including fibronectin, vitronectin, osteopontin, and tenascin-C (18–20). These proteins were thus surveyed for expression in the cardiac vasculature and subsequent co-localization with ψR3Y32. Immunostaining for the phage-coat protein, pIII, demonstrated expression within the cardiac vasculature of ψR3Y32injected mice, confirming the binding of ψR3Y32 to vessels within the heart (Fig. 1B). TenascinC expression was also observed within the cardiac vasculature, most notably associated with venules, but also within the capillary network and in arterioles (Fig. 1B–D). Tenascin-C expression was found to co-localize with pIII in the heart, suggesting that ψR3Y32 mimics an endogenous binding partner for tenascin-C in the cardiac vasculature. Co-staining for tenascin-C and the nuclear marker DAPI further confirmed the endovascular localization of tenascin-C in cardiac vessels (Fig. 1D). Additionally, expression of tenascin-C was detected in the bone marrow sinusoidal vasculature (Fig. 1E). Fibronectin expression also co-localized with phage immunostains in the cardiac vasculature of the heart (Fig. 1F, G). In the bone marrow, fibronectin expression has previously been reported in the vasculature (21). This protein therefore represented another candidate that may be


involved in modulation of pro-angiogenic pathways in both the heart and bone marrow. Osteopontin and vitronectin were not identified in the murine heart (data not shown). As such, tenascin-C and fibronectin alone were further analyzed for potential roles in the promotion of pro-angiogenic pathways. Tenascin-C regulates cardiac endothelial cell adhesion in a temporally dynamic manner The preferential binding of ψR3Y32 in the young heart as well as the immunohistochemical data led us to hypothesize that tenascin-C and/or fibronectin regulate cardiac angiogenic mechanisms. We therefore sought to determine the role of these proteins in regulating CMEC phenotype and behavior. CMECs cultured on tenascin-C revealed temporal differences in adhesion compared with those plated on either fibronectin or collagen. After 3 h of culture, cells cultured on collagen and fibronectin had attached well and were beginning to spread (Fig. 2A, B). Cells grown on tenascin-C, however, retained a highly spherical morphology at this time-point (Fig. 2C). After 24 h, cells on tenascin-C resembled those cultured on collagen or fibronectin and had become well attached and spread to form a typical endothelial monolayer (Fig. 2D–F). This action of tenascin-C appears to be cell type-specific, since human umbilical vein endothelial cells (HUVECs) cultured on tenascin-C in the same manner are well spread and attached within 3 h of plating (data not shown), as has been previously demonstrated (22). Based on the differential phenotype of CMECs on tenascin-C, compared with collagen and fibronectin, the potential proangiogenic actions of tenascin-C were further investigated. To compare the affinity of the cell-matrix interactions of the spherical cells on tenascin-C with those exhibiting a flattened morphology, we plated cells on tenascin-C and collagen (as a control) and determined the number of cells that failed to attach after 10 min and 3 h. Within 10 min of plating, while 83% of cells had become well attached to the collagen substrate, strikingly, 89% of cells failed to attach to tenascin-C within this time (Fig. 2E). After 3 h of culture, 99% of cells had attached to the collagen substrate, while approximately 10% of cells cultured on tenascin-C had still failed to adhere. Thus, tenascin-C mediates CMEC attachment in a temporally dynamic manner and, at least within the first few hours of CMEC binding to tenascinC, maintains cells with a spherical phenotype with delayed adhesive properties. Tenascin-C promotes cardiac endothelial cell migration in response to angiogenic growth factors In order for fully differentiated endothelial cells to contribute to vascular remodeling after ischemic damage, these cells must undergo a pro-angiogenic activation, disrupting their cellextracellular matrix (ECM) interactions and altering their morphology. Based on the early antiadhesive action of tenascin-C upon interaction with CMECs, we hypothesized that this may function as an angiogenic inducer to enhance the migratory capacity of these cells. To test this, we developed a 3-dimensional assay system in which CMECs were cultured on either collagen or tenascin-C for 3 h, and subsequent migration through a collagen gel was analyzed in response to PDGF-AB, VEGF-A, or no angiogenic growth factors, as a control (Fig. 3A, B). After 48 h, peak vertical migration distances through the z-axis of the gel were quantified (Fig. 3C). This analysis revealed that, while CMECs can migrate from both tenascin-C and collagen in the absence of stimulatory growth factors, migration is greatly enhanced in the presence of either PDGF-AB or VEGF-A. Peak migration distance is increased for cells cultured on collagen by 51


and 63% in response to PDGF and VEGF, respectively, compared with cells cultured on collagen in the absence of these chemotactic growth factors. Strikingly, cells cultured on tenascin-C display migration rates that are even further enhanced in response to these growth factors (64 and 92% respectively), demonstrating that interaction of CMECs with tenascin-C specifically promoted the migration of the cells in response to angiogenic signals. Endothelial progenitor cells incorporate at sites of tenascin-C expression in the heart We have previously demonstrated that PDGF signaling is important for postnatal cardiac remodeling, promoting angiogenesis mediated by a subpopulation of PDGFRα-positive endothelial cells and bone marrow-derived EPCs (11, 23, 24). Furthermore, tenascin-C is known to be induced by PDGF in other vascular cell types (11, 25). These data, therefore, suggested that tenascin-C might act as a downstream component of PDGF-mediated angiogenic mechanisms. Indeed, in vitro RT-PCR analysis of tenascin-C expression in CMECs cultured with PDGF revealed an up-regulation of tenascin-C mRNA, peaking at 6 h of PDGF-AB treatment (>6-fold increase; Fig. 4A, B). Additionally, 24 h after intramyocardial injection of PDGF-AB, PDGFRαpositive cells were localized at sites of tenascin-C protein expression in the cardiac vasculature (Fig. 4C, D). Together these data suggest that tenascin-C can act downstream of PDGF-AB and might regulate both local endothelial cell function as well as EPC incorporation. To further examine the association of tenascin-C with EPCs in the heart, genetically tagged ROSA-26 (βgal+) bone marrow cells were injected systemically into irradiated young mice, which was followed 1 month later by intramyocardial administration of PDGF-AB. This resulted in donorderived (β-gal+) cell recruitment to the heart within 24 h of PDGF-AB treatment, with the majority of donor cells incorporating at sites of tenascin-C protein expression (Fig. 4E, F). Thus, tenascin-C is associated with sites of EPC integration into the cardiac vasculature, suggesting that this protein may regulate both local endothelial function as well as EPC homing and/or incorporation at sites of angiogenic induction. Tenascin-C is localized to neovascular channels in human coronary thrombi Given the co-localization of tenascin-C with sites of EPC incorporation and local endothelial activation in the rodent heart, we hypothesized that tenascin-C would also be associated with sites of cardiac vascular remodeling in human cardiopathologies. Based on the endovascular patterning of tenascin-C and the reported role of EPCs in channel formation in intravascular thrombi (26), we examined human cardiac thrombi extracted by percutaneous intracoronary thrombectomy from patients with acute coronary syndromes (Fig. 5A–C). Immunostains revealed that tenascin-C is indeed present within these thrombi (Fig. 5D–F) and, moreover, is co-localized with the endothelial/EPC marker Tie-2 (Fig. 5G–I). Importantly, this is the first identification of tenascin-C in coronary thrombi and its localization in the vasculature supports a role for this protein in recanalization of fibrin clots via neovascularization. Tenascin-C is required for fibrin vascularization in murine cardiac transplants To investigate the importance of tenascin-C in fibrin neovascularization, we used an in vivo murine model. While pro-coagulant manipulations, either chemical or mechanical, have been used to induce thrombus formation in different vascular beds (27), these have not been used to specifically promote fibrin deposition in murine coronary arteries. We, therefore, chose to use a


syngeneic cardiac allograft model in which neonatal cardiac tissue is transplanted into a subdermal tissue pocket in the pinna of a syngeneic host (11, 12). In this model, a fibrin clot forms around the allograft. This clot, as well as the cardiac tissue itself, is then vascularized by a combination of host endothelial cells and bone marrow-derived EPCs. Wild-type neonatal hearts were transplanted into the pinnae of young wild-type or tenascin-C–/– mice. Three days after transplantation, the peri-allograft region of wild-type host mice revealed formation of a fibrin clot. Moreover, the fibrin of the thrombus was infiltrated by vascular channels that were positive for tenascin-C protein and were often associated with Tie-2-positive cells (Fig. 6B–D). Analysis of tenascin-C–/–- host mice at 3 days also showed significant fibrin deposition. However, no vascular channels were present in these clots (Fig. 6E, F). These data therefore confirm that tenascin-C is essential for fibrin vascularization. Tenascin-C knockout mice have impaired cardiac angiogenic mechanisms Given the deficiency in fibrin neovascularization observed 3 days after cardiac allograft transplantation, we hypothesized that a lack of tenascin-C would inhibit subsequent vascularization of the cardiac allografts. Analysis of wild-type mice 7 days post-transplantation demonstrated that fibrin had resolved and the transplanted cardiac tissue was viable, as determined by visual assessment and ECG. Allografts transplanted into tenascin-C–/– mice, however, were not viable and evidence of thrombus at the engraftment site remained (Fig. 7A, B). This provides further evidence that tenascin-C is important in postnatal vascular remodeling. Work from our group and others has previously shown that bone marrow-derived EPCs contribute to cardiac angiogenesis (12, 28, 29). We have demonstrated that transplantation of cardiac allografts into 18-month-old mice results in loss of the hearts as well as the surrounding pinnal tissue. Importantly, this phenotype is reversed by the prior transplantation of bone marrow cells from young (3-month-old), but not old, mice (12). To assess whether the actions of tenascin-C are essential for bone marrow cell-mediated cardiac angiogenesis, we performed bone marrow transplantation from young wild-type or tenascin-C–/– mice into aging hosts 1 week prior to neonatal cardiac transplantation. Consistent with previous findings, young wild-type bone marrow cells transplanted into intact aging hosts restored cardiac angiogenic function that is dysregulated with age (Fig. 7A–C). Transplantation of bone marrow from young tenascin-C–/– mice, however, failed to restore this pathway, demonstrating that tenascin-C is essential for bone marrow cell-mediated mechanisms of postnatal neovascularization (Fig. 7A, B). Histological examination demonstrated that, as with wild-type donor bone marrow, tenascin-C–/– donor bone marrow cells were able to colonize the host bone marrow. However, a paucity of these cells in the periallograft region, compared with controls, suggests that a lack of tenascin-C in the bone marrow results in impaired mobilization and/or homing to the site of vascular remodeling (Fig. 7C, D). DISCUSSION The present study has identified tenascin-C as an important mediator of cardiac angiogenic mechanisms. We have demonstrated for the first time that tenascin-C is specifically localized to the vasculature of the heart, notably at sites of EPC incorporation and local PDGFRα-positive endothelial cell recruitment, and, importantly, at sites of neovascularization after coronary thrombus and fibrin formation. In vitro, tenascin-C promotes CMEC de-adhesion and subsequent chemotactic migration, which suggests a potential role in the early stages of local angiogenic


induction. Significantly, our findings also represent the first evidence that tenascin-C is essential for postnatal cardiac vascular remodeling, specifically promoting fibrin neovascularization and cardiac tissue angiogenesis. Tenascin-C has been implicated as playing a role in angiogenesis in a variety of models, based on immunohistochemical localization at sites of vascular remodeling and in vitro assays of endothelial function in the presence of tenascin-C. Tenascin-C expression is highly associated with angiogenesis in a wide range of disease states, for example, including cancer, diabetes, and aortic aneurysm (30–32). In vitro, tenascin-C is specifically expressed by sprouting and cordforming aortic endothelial cells but not by nonsprouting cells (33) and is up-regulated in pericyte/endothelial cord formation (34). In the central nervous system, tenascin-C is expressed by migrating endothelial cells and the kinetics of its expression are linked with maximal angiogenic activity (35). Despite this wealth of correlative evidence, the tenascin-C–/– mouse has not been shown to have any overt vascular phenotypic defects (36). Furthermore, the role of tenascin-C in postnatal cardiac angiogenesis has not previously been defined. The identification of tenascin-C within the vasculature of the heart suggests that it may have a role in the intravascular neovascularization of thrombi caused by plaque rupture in acute coronary syndromes. Indeed, the finding that tenascin-C is expressed in the dynamic vascular plexus that forms in coronary thrombi in humans, as well as in our rodent cardiac transplantation model, supports a role for this protein in cardiac neovascularization. While the colocalization of tenascin-C with Tie-2 is consistent with its proangiogenic actions, we cannot determine from our immunohistochemical analysis whether tenascin-C interacts primarily with local endothelial cells or EPCs in the fibrin, since Tie-2 is a marker of both cell types. It has been postulated recently, however, that while EPCs home to thrombi in mice, they do not contribute to the vasculature but instead provide support via secretion of angiogenic growth factors (6, 26). The results of the murine transplantation studies establish that tenascin-C is essential for fibrin neovascularization and cardiac angiogenesis, which suggests that the actions of tenascin-C in the coronary thrombi are important. Previous studies have demonstrated that tenascin-C is present at sites of coronary atheroma and it has been speculated that tenascin-C may induce coronary plaque formation (37). Based on its importance in angiogenesis, we suggest that this link might be due to the promotion of vascular remodeling pathways in the atherosclerotic lesion. Moreover, tenascin-C has been shown to be up-regulated in the heart after myocardial infarction (38). Since the viability of cardiac transplantation in mice inversely correlates with the extent of myocardial infarction in rat models of cardiac ischemia (11), our findings further support a role for tenascin-C in tissue repair and formation of a collateral blood supply after vascular damage. Cardiovascular repair mechanisms are compromised in the aging heart but can be restored by PDGF-AB, which increases angiogenesis and limits the extent of myocardial infarction-related injury (11). The co-localization of tenascin-C with PDGFRα-positive cells and the PDGF-ABinduced up-regulation of tenascin-C by CMECs suggests that tenascin-C may be a downstream component of PDGF-AB-mediated cardioprotective mechanisms. Its precise role in this process is unclear but, based on our data, we hypothesize that up-regulation of PDGF-AB expression in response to vascular injury and platelet activation results in increased tenascin-C expression,


which in turn provides a platform for EPCs as they home to sites of damage, and/or local endothelial cells that must undergo angiogenic induction to contribute to remodeling vessels. The mechanisms mediating the actions of tenascin-C on angiogenesis and fibrin neovascularization may be organ- and cell type-specific. In fact, much investigation has focused on the specific actions of tenascin-C on a variety of cell types, often revealing seemingly contradictory effects. Tenascin-C promotes attachment of HUVECs and fibroblasts in vitro, for example (22, 39), but appears to be anti-adhesive for oligodendrocytes and neural crest cells (39– 41). Similarly, tenascin-C promotes migration of HUVECs and bovine retinal endothelial (22, 31) but inhibits migration of glioma cells and oligodendrocytes (42, 43). Since the actions of tenascin-C are cell type-specific, the dynamic, anti-adhesive function of tenascin-C on the cardiac endothelium may be central to its role in cardiac angiogenesis and neovascularization. De-adhesion has been proposed to be a step in conversion from a quiescent state to one in which a cell is responsive to local injury (44). The adhesive and morphological changes observed in CMECs are consistent with our data, which demonstrate enhanced migration of CMECs upon association with tenascin-C. These in vitro findings, however, may not directly correlate with the actions of tenascin-C in vivo, since CMECs in their native environment do not interact with tenascin-C alone but are exposed to a wide variety of ECM molecules, including fibronectin and collagen, all of which may exert actions that modulate the adhesive and migratory properties of this cell type. Indeed, the dynamic association of CMECs, as well as EPCs, with tenascin-C is likely to be under tight regulation, maintaining a balance between endothelial cells in a differentiated, quiescent state and a pro-angiogenic phenotype, which promotes cell migration in conditions of vascular injury, such as myocardial infarction or thrombus formation. Such regulation likely involves a number of factors, including induction by PDGF-AB secreted by aggregated platelets (45), variation in the isoforms of tenascin-C expressed, and enzymatic processing of these isoforms by proteinases such as the matrix metalloproteinases (46). The expression of other ECM molecules by CMECs after contact with tenascin-C is also likely to be a major factor regulating phenotypic changes. In particular, our immunohistochemical data suggest that the balance between fibronectin and tenascin-C expression might dictate whether cells are well attached or disadhesive. It has been demonstrated that tenascin-C and fibronectin expression are often concomitant and the lack of tenascin-C expression in the tenascin-C–/– mouse results in a significant decrease in fibronectin expression during wound healing (47). Fibronectin alone is adhesive for CMECs, therefore, after initial contact with tenascin-C, cells may begin to express fibronectin, shifting the balance from an anti-adhesive to a pro-adhesive state. Thus, tenascin-C and fibronectin may work in concert to facilitate in vivo mechanisms of neovascularization via endothelial cells and, potentially, EPCs. The finding that bone marrow cells from tenascin-C–/– mice fail to restore cardiac angiogenic mechanisms in the aging host provides definitive evidence that disruption of tenascin-C expression in the bone marrow population alone is sufficient to impair cardiac neovascularization. While a role for bone marrow-derived EPCs has been demonstrated in many animal models of vascular injury (9, 28, 29), the present study and previous work from our lab (11) conclusively show that bone marrow cells play a crucial rather than supplementary role in vascular repair mechanisms. Bone marrow tenascin-C expression has been shown to regulate the generation of hematopoietic stem cells, and the tenascin-C–/– mouse has age-associated defects in hematopoietic activity (48). Since hematopoietic stem cells and EPCs are thought to share a


common lineage and utilize common mechanisms of mobilization (49), our data are consistent with these previous findings and suggest that tenascin-C regulates the activity of EPCs as well as hematopoietic cells in the bone marrow. The lack of tenascin-C–/– bone marrow cells at the site of cardiac allograft vascularization further supports the importance of tenascin-C in EPCmediated angiogenesis. There has been little analysis of changes in tenascin-C expression with age, and this is the first evidence of a bone marrow cell-specific defect associated with neovascularization in the tenascin-C–/– mouse. We have observed a down-regulation in tenascinC expression in the aging bone marrow (unpublished observations), which may account, at least in part, for the age-associated decrease in EPC mobilization from the bone marrow (50). Thus, tenascin-C likely plays a significant role in the impairment in vascular repair pathways that takes place with increasing age (11). Together these findings point to a novel role for tenascin-C as a regulator of both systemic and local mechanisms of cardiac neovascularization. Clinically, antibodies directed against tenascinC inhibit tumor angiogenesis (51). Our data suggest that promotion of tenascin-C expression may conversely enhance angiogenic cardioprotection and repair mechanisms. This protein may therefore represent a novel target for induction or restoration of angiogenic pathways that are down-regulated with age and disease. ACKNOWLEDGMENTS J.M.E. is a Charles E. Culpepper medical scholar. This work was supported in part by the Rockefeller Brothers fund and by the NIH (AG19738, AG20320, AG20918, HL67839). The authors wish to thank Dr. Donna Whitlon (Northwestern University) and Dr. Moriaki Kusakabe (Jikei University, Japan) for providing the tenascin-C–/– mice. REFERENCES 1.

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Fig. 1

Figure 1. ψR3Y32 binding in young and old bone marrow and hearts. A) Phage ψR3Y32 variable region epitope with

sequence homology to extracellular domain of α8-integrin. Quantification of ψR3Y32 binding in young and old bone marrow and hearts (n=4 per group). *P < 0.001. B) Co-immunostaining of tenascin-C (red) and phage coat protein pIII (green) in sections of the young rodent heart, demonstrating binding of ψR3Y32 to areas of tenascin-C expression in cardiac venules and microvessels. Insets illustrate separate signals for tenascin-C and pIII immunostains and negative control with no primary antibodies. C) Tenascin-C expression in a venule of the heart. D) Tenascin-C and DAPI co-stain demonstrate luminal expression of tenascin-C in an arteriole. E) Tenascin-C in bone marrow vasculature (arrowheads). Inset, negative control. F) Co-immunostaining of fibronectin (red) and phage coat protein pIII (green) in sections of the young rodent heart (arrows). Insets, separate signals for fibronectin and pIII immunostains. G) Fibronectin expression in a venule of the heart (arrows). Scale bars in (B), (D), (E), (F), and (G), 50 µm; in (C) and inset in (E), 100 µm.


Fig. 2

Figure 2. Cardiac microvascular endothelial cells display diminished attachment and spreading on tenascin-C at early, but not late time-points. Rat CMECs were cultured on 10 µg/ml collagen (A, D), fibronectin (B, E), or tenascin-C (C, F) for 3 h (A–C) or 24 h (D–F). While cells plated on collagen and fibronectin readily attach and form a monolayer, this process is delayed in cells cultured on tenascin-C. G) Cell adhesion analysis after plating cardiac endothelial cells on collagen or tenascin-C (n=4–8 per group). *P < 0.0001.


Fig. 3

Figure 3. Cardiac microvascular endothelial cells demonstrate increased migratory activity when cultured on tenascin-C. A) CMECs were cultured on tenascin-C or collagen for 3 h prior to overlaying of a collagen gel matrix and addition of angiogenic growth factors to the top of the gel. B) After 48 h, cell migration could be seen through the gel. Arrows, cells in plane of focus in collagen gel. Arrowheads, cells above or below plane of focus, indicating vertical migration through gel. C) Quantification of peak migratory distance, based on distance traveled by top 5 cells in each well, revealed that cells cultured on tenascin-C migrate further than those on collagen, in response to both PDGF-AB and VEGF-A (n=8 per group). *P < 0.05.


Fig. 4

Figure 4. Endothelial progenitor cells incorporate at sites of tenascin-C expression in the heart. A) RT-PCR analysis of tenascin-C mRNA expression after PDGF treatment of cardiac microvascular endothelial cells in vitro. Representative gel illustrates peak induction of tenascin-C expression after 6 h of PDGF-AB treatment. B) Real-time RT-PCR analysis of induction of tenascin-C mRNA expression after 6h of PBS (control) vs. PDGF-AB treatment (n=3 per group). C, D) Representative examples of co-localization of tenascin-C (red) and PDGFRα (green) expression (arrows) in venules of young PDGF-AB-treated mouse hearts. Lower left inset in (C), negative control (i.e., no primary antibody); insets at top and bottom right side of each panel illustrate separate signals for tenascin-C and PDGFRα expression, respectively. E, F) Representative examples of tenascin-C (red) and β-gal (green) colocalization in young PDGF-treated mouse hearts sacrificed 24 h after tail vein injection of whole β-gal-positive bone marrow from a young ROSA-26 mouse. Lower left inset in (E), negative control; insets at top and bottom right side of each panel illustrate separate signals for tenascin-C and β-gal expression, respectively. Scale bars in (C) and (E), 50 µm, (D) and (F), 100 µm.


Fig. 5

Figure 5. Endothelial cells in human coronary thrombi express tenascin-C. A) Angiogram demonstrating an occlusion in the proximal left circumflex artery in an individual with acute coronary syndrome (dashed line-course of occluded vessel). B) Angiogram after percutaneous thrombectomy. C) Gross specimen of the coronary thrombi after extraction and fixation. Tenascin-C protein immunostaining is found in large and small channels (D–G) in the coronary thrombi. This patterning co-localizes with the endothelial marker, Tie-2 (G). Purple immunostains in (D) and (G), peroxidase labeling of primary anti-tenascin-C antibody and visualization with Vector VIP; scale bar in (C), 2 mm; in (D), 50 µm; in (E), 100 µm; in (F) and (G), 10 µm. Inset in (E), negative control. Arrowheads in (F), nuclei (blue, DAPI) surrounded by tenascin-C expression (green). Tn-C, tenascin-C.


Fig. 6

Figure 6. Tenascin-C –/– mice fail to vascularize cardiac thrombi. A) Model of cardiac allograft transplantation. Neonatal wild-type hearts are inserted into the pinnae of young wild-type or tenascin-C –/– mice. After 7 days, viability is scored based on visual assessment of healthy cardiac allograft and lack of thrombosis (B, C), as well as ECG activity of the allograft (C; n≥6 transplants per group). Three days after transplantation, Giemsa-stained sections of the pinnae of wildtype mice display fibrin vascularization (E, F). Immunostaining reveals that these vessels are positive for tenascin-C (brown signal in upper and lower left insets in (G); green signal in right inset in (G)) and are associated with Tie-2-positive [red signal in right inset in (G)] cells. In contrast, sections of allografts in tenascin-C–/– mice display evidence of thrombus formation, but with no associated vascularization (F, G). Yellow arrows in (E) and (H), transplanted heart tissue; black arrows in (E) and (H), fibrin of thrombus; asterisks in (E) and (H) denote regions of each section shown at higher magnification in (F) and (I), respectively; arrowheads in (F) delineate a vascular channel penetrating the fibrin clot; yellow arrows in (G), DAB-positive tenascin-C signal. Scale bars in (E) and (H), 250 µm, in (F) and (I), 100 and 50 µm, respectively.


Fig. 7

Figure 7. Tenascin-C –/– mice have impaired cardiac angiogenic capacity. A) Representative images and electrocardiograms of cardiac allografts transplanted into the pinnae of 3- and 18-month-old hosts (either wild-type (WT) or tenascin-C–/–), with or without prior transplantation of 3-month-old bone marrow from wild-type (C57Bl/6) or tenascin C–/– mice into intact, unirradiated hosts. B) Allograft viability 7 days after transplantation (n=12–43 per group). C) Section of pinna from old host receiving young ROSA-26 bone marrow transplantation (peri-allograft region). Blue cells, β-gal+ donor bone marrow cells. Inset, host bone marrow colonized by donor β-gal+ donor cells. D) Section of pinna from old host receiving young tenascin-C–/– bone marrow. Note lack of β-gal+ donor cells. Inset, host bone marrow colonized by donor β-gal+ tenascin-C–/– cells. Arrows in (A), intact, vascularized cardiac allograft; white arrowheads in (A), cardiac allograft surrounded by fibrin clot, black arrowheads in (A), necrotic loss of allograft and host pinnal tissue; arrows in (D), blood clot at site of necrotic loss of allograft. Scale bars in (C) and (D), 50 μm, P < 0.05.
