blood vessel walls. PMSC have a mixed phenotype of mesenchymal stem cells (MSC): CD73, CD90, CD105 and pericyte cells (PC): NG2, CD146. CD140b.
Human Perivascular Mesenchymal Stem Cells: Tissue-Resident Regulators of Vessel Wall Homeostasis.
Krzysztof Korta1, Piotr Kupczyk2,3, Jakub Grzesiak4, Aleksandra Piotrowska5, Maria Paprocka6, Marcin Hołysz7, Roksana Jura8, Ela Piątkowska8, Katarzyna Mikołajewicz2, Grzegorz Chodaczek2, Piotr Dzięgiel5, Beata Nowakowska✝3, Jan Skóra1. 1. Department and Clinic of Vascular, General and Transplantation Surgery, Jan-Mikulicz Radecki University Hospital, Silesian Piast Wrocław Medical University, Borowska 213 Street, 50-556 Wrocław, Poland. 2. Laboratory of Confocal Microscopy, Department of Biotechnology, Wrocław Research Centre EIT+, Stabłowicka 147 Street, 54-066 Wrocław, Poland. 3. Laboratory of Immunogenetic and Tissue Immunology, Department of Clinical Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, Weigla 12 Street, 53-114 Wrocław, Poland. 4. Laboratory of Electron Microscopy, Department of Biotechnology, Wrocław Research Centre EIT+, Stabłowicka 147 Street, 54-066 Wroclaw, Poland. 5. Department of Histology and Embryology, Silesian Piast Wrocław Medical University, Chałubińskiego 6a Street, 50-368 Wrocław, Poland. 6. Laboratory of Glycobiology and Cellular Interactions, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, Weigla 12 Street, 53-114 Wrocław, Poland. 7. Department of Biochemistry and Molecular Biology, Karol Marcinkowski University of Medical Sciences, Święcickiego 6 Street, 60-781 Poznań, Poland. 8. Laboratory of Mass Spectrometry, Department of Biotechnology, Wrocław Research Centre EIT+, Stabłowicka 147 Street, 54-066 Wrocław, Poland.
Introduction: Perivascular Mesenchymal Stem Cells (PMSC) are a subgroup of Vascular Stem Cells (VSC) which reside at quiescent state within niches of large blood vessel walls. PMSC have a mixed phenotype of mesenchymal stem cells (MSC): CD73, CD90, CD105 and pericyte cells (PC): NG2, CD146 CD140b. PMSC as cells of mesodermal origin exhibit multipotential properties, express HLA-ABC in the absence of HLA-DR antigens, while in culture, are heterogeneous adherent fibroblast-like cells. PC residing in large-diameter vessel walls with three-layer biostructure (tunica: intima, media and adventitia) are found in two locations: directly neighborhood of tunica intima endothelial cells (EC) and microvascular network of vessels walls - vasa vasorum (VV). The latter is considered to be a residence of PMSC and is formed by a network of arterioles and venules comprising EC surrounded by PC. The quiescent state of PMSC is regulated by their tissue environment, which is equivalent to the canonical hematopoietic stem cell (HSC) niche. PC focused much attention of research community because they present immunosuppressive or regenerative properties, similarly to MSC. For this reason there have been even speculation that PC are tissue-specific VSC precursors and counterparts for MSC, but this intriguing question remains still unanswered. PMSC residing within tunica media and adventitia may participate in vessel wall homeostasis. It cannot by excluded that their dysfunctions may be the molecular basis of cardiovascular diseases (CVD). Material and methods:
Vessel wall segments of vena saphena were collected during ambulatory phlebectomy proceedings. Vessels were rinsed thoroughly, cut into pieces and put to 4% buffered PFA for immunohistochemistry or immediately placed in DMEM with 20% FBS and 1.5% penicillin-streptomycin antibiotic for cell culturing. PMSC distribution in vessel wall was analyzed by immunostaining using monoclonal NG2 antibody (Abcam, UK) with DAB detection system (DAKO, Denmark). Vessel wall intima, media and adventitia layers were enzymatically separated using tissue-dedicated compound Liberase TH (Roche, USA) for 2 hours. After mechanical separation of tunica media from adventitia layers were digested overnight with Liberase TM. Obtained cells were filtered and seeded into culture dishes in Pericyte Growth Medium (Promocell, Germany). Gene and protein expression analysis was performed when cells became adherent, had expected morphology and reached confluency. MSC and PC transcripts were detected upon RNA isolation (Qiagen, Germany) followed by reverse transcription (Roche, USA) real-time PCR with UPL probes. The protein expression analysis was performed on 4% PFA fixed cells by confocal microscopy and flow cytometry.
Results: Basing on histological (H&E) analysis only vessel walls with absence of pathomorfological vessel-tissue remodeling were classified for next step of the study (Fig.1: a, b). Immunohistochemical analysis show NG2+ cells in medial and adventitial microvascular network of VV (Fig.2: a, b). After vessel digestion, cell isolation and seven days of culture gene expression analysis show high expression transcripts for MSC and PC respectively, but low level of CD34 and absence of CD31 (data not shown). Light and confocal microscopy analysis show that PMSC are heterogeneous population of adhesive cells with typical morphology of fibroblast-like cells. In vitro confocal analysis show high expression of CD105, CD140b, SMA by most of the cells (Fig.3: a, b and Fig.4: a, b), CD90, CD146 and NG2 were restricted to some cells, while CD34 was low and CD31 was completely absence (data not sown). We show that all cells express adhesion molecules: Ninj-1/2, neurovascular molecules characteristic for tissue injury, but also active capillary PC (Fig.5: a, b). Flow cytometry analysis show especially high expression of MSC markers: CD90, CD73 and CD105, while lower expression of CD140a, CD140b and minimal expression of NG2, CD146 (data not shown) may reflect perivascular origin of PMSC. Flow cytometry analysis exhibit high expression of major HLA-ABC antigen in absence of HLA-DR minor antigens. Fig.1
a Fig.2
H&E-x20
H&E-x100
b
a Fig.3
NG2-x20
NG2-x100
b
a Fig.4
NG2+CD140b+DAPI
CD146+CD140b+DAPI
a Fig.5
CD90+CD140b+DAPI
CD105+SMA+DAPI
a
CD73
CD140a
HLA-ABC
CD90
CD140b
HLA-DR
CD105
CD34
CD31
NG2+Ninj-1+DAPI
NG2+Ninj-2+DAPI
Conclusions: We identified mesodermal cells within vessel wall with mixed phenotype of MSC and PC. Our data show that PMSC are perivasculary localized cells, which in culture are heterogenous, adherent and fibroblast-like cells with morphology and phenotype closely related to MSC. In next step of our investigations we are going to confirm, if PMSC may regulate local regeneration or immune functions and participating in vessel wall homeostasis.
Wroclaw Medical University Grant: Resident Vascular Stem and Progenitor Cells in Biostructure of Blood Vessel Walls.
