Secreted Protein Acidic and Cysteine Rich Matricellular Protein is Enriched in the Bioactive Fraction of the Human Vascular Pericyte Secretome.

Aims: To ascertain if human pericytes produce SPARC (acronym for Secreted Protein Acidic and Cysteine Rich), a matricellular protein implicated in the regulation of cell proliferation, migration, and cell-matrix interactions; clarify if SPARC expression in cardiac pericytes is modulated by hypoxia; and determine the functional consequences of SPARC silencing. Results: Starting from the recognition that the conditioned media (CM) of human pericytes promote proliferation and migration of cardiac stromal cells, we screened candidate mediators by mass-spectrometry analysis. Of the 14 high-confidence proteins (<1% FDR) identified in the bioactive fractions of the pericyte CM, SPARC emerged as the top-scored matricellular protein. SPARC expression was validated using ELISA and found to be upregulated by hypoxia/starvation in pericytes that express platelet-derived growth factor receptor α (PDGFRα). This subfraction is acknowledged to play a key role in extracellular matrix remodeling. Studies in patients with acute myocardial infarction showed that peripheral blood SPARC correlates with the levels of creatine kinase Mb, a marker of cardiac damage. Immunohistochemistry analyses of infarcted hearts revealed that SPARC is expressed in vascular and interstitial cells. Silencing of SPARC reduced the pericyte ability to secrete collagen1a1, without inhibiting the effects of CM on cardiac and endothelial cells. These data indicate that SPARC is enriched in the bioactive fraction of the pericyte CM, is induced by hypoxia and ischemia, and is essential for pericyte ability to produce collagen. Innovation: This study newly indicates that pericytes are a source of the matricellular protein SPARC. Conclusion: Modulation of SPARC production by pericytes may have potential implications for postinfarct healing.

MicroRNA-532-5p Regulates Pericyte Function by Targeting the Transcription Regulator BACH1 and Angiopoietin-1.

MicroRNAs regulate endothelial function and angiogenesis, but their implication in pericyte biology remains undetermined. A PCR array, covering a panel of 379 human microRNAs, showed microRNA-532-5p to be one of the most differentially modulated by hypoxia, which was confirmed by qPCR in both skeletal muscle and adventitial pericytes. Furthermore, microRNA-532-5p was upregulated in murine muscular pericytes early after experimentally induced ischemia, decreasing below baseline after reperfusion. Transfection of human pericytes with anti-microRNA, microRNA-mimic, or controls indicates microRNA-532-5p modulates pro-angiogenic activity via transcriptional regulation of angiopoietin-1. Tie-2 blockade abrogated the ability of microRNA-532-5p-overexpressing pericytes to promote endothelial network formation in vitro. However, angiopoietin-1 is not a direct target of microRNA-532-5p. In silico analysis of microRNA-532-5p inhibitory targets associated with angiopoietin-1 transcription indicated three potential candidates, BACH1, HIF1AN, and EGLN1. Binding of microRNA-532-5p to the BACH1 3' UTR was confirmed by luciferase assay. MicroRNA-532-5p silencing increased BACH1, while a microRNA-532-5p mimic decreased expression. Silencing of BACH1 modulated angiopoietin-1 gene and protein expression. ChIP confirmed BACH1 transcriptional regulation of angiopoietin-1 promoter. Finally, microRNA-532-5p overexpression increased pericyte coverage in an in vivo Matrigel assay, suggesting its role in vascular maturation. This study provides a new mechanistic understanding of the transcriptional program orchestrating angiopoietin-1/Tie-2 signaling in human pericytes.

The adipokine leptin modulates adventitial pericyte functions by autocrine and paracrine signalling.

Transplantation of adventitial pericytes (APCs) improves recovery from tissue ischemia in preclinical animal models by still unknown mechanisms. This study investigates the role of the adipokine leptin (LEP) in the regulation of human APC biological functions. Transcriptomic analysis of APCs showed components of the LEP signalling pathway are modulated by hypoxia. Kinetic studies indicate cultured APCs release high amounts of immunoreactive LEP following exposure to hypoxia, continuing upon return to normoxia. Secreted LEP activates an autocrine/paracrine loop through binding to the LEP receptor (LEPR) and induction of STAT3 phosphorylation. Titration studies using recombinant LEP and siRNA knockdown of LEP or LEPR demonstrate the adipokine exerts important regulatory roles in APC growth, survival, migration and promotion of endothelial network formation. Heterogeneity in LEP expression and secretion may influence the reparative proficiency of APC therapy. Accordingly, the levels of LEP secretion predict the microvascular outcome of APCs transplantation in a mouse limb ischemia model. Moreover, we found that the expression of the Lepr gene is upregulated on resident vascular cells from murine ischemic muscles, thus providing a permissive milieu to transplanted LEP-expressing APCs. Results highlight a new mechanism responsible for APC adaptation to hypoxia and instrumental to vascular repair.

Vascular stem cells-potential for clinical application.

INTRODUCTION: Cell therapy is a growing area of research as an alternative to pharmaceuticals or surgery for the treatment of ischaemic disease. Studies are focusing on delivering tissue-derived cells into damaged organs to promote vascular regeneration or gain of function. SOURCES OF DATA: Pubmed, clinicaltrials.gov, BHF website. AREAS OF AGREEMENT: Stem cells have the potential to become a viable treatment for many diseases, as indicated by the numerous pre-clinical studies demonstrating therapeutic benefit. AREAS OF CONTROVERSY: The mechanisms of action for transplanted stem cells are still open to debate. Proposed mechanism includes direct cell incorporation and paracrine action. Additionally, the secretome produced by transplanted cells remains largely unknown. GROWING POINTS: Initial studies focused on delivering stem cells by injection; however, current research is utilizing biomaterials to target cell delivery to specific areas. AREAS TIMELY FOR DEVELOPING RESEARCH: Whilst stem cell research in the laboratory is expanding rapidly, transition into clinical studies is hindered by the availability of equivalent clinical grade reagents.

Expansion and characterization of neonatal cardiac pericytes provides a novel cellular option for tissue engineering in congenital heart disease.

BACKGROUND: Living grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts. METHODS AND RESULTS: CD34(pos) cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34(pos)/CD31(neg) cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kit(pos) cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft. CONCLUSIONS: These findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.

Epigenetic profile of human adventitial progenitor cells correlates with therapeutic outcomes in a mouse model of limb ischemia.

OBJECTIVE: We investigated the association between the functional, epigenetic, and expressional profile of human adventitial progenitor cells (APCs) and therapeutic activity in a model of limb ischemia. APPROACH AND RESULTS: Antigenic and functional features were analyzed throughout passaging in 15 saphenous vein (SV)-derived APC lines, of which 10 from SV leftovers of coronary artery bypass graft surgery and 5 from varicose SV removal. Moreover, 5 SV-APC lines were transplanted (8×10(5) cells, IM) in mice with limb ischemia. Blood flow and capillary and arteriole density were correlated with functional characteristics and DNA methylation/expressional markers of transplanted cells. We report successful expansion of tested lines, which reached the therapeutic target of 30 to 50 million cells in ≈10 weeks. Typical antigenic profile, viability, and migratory and proangiogenic activities were conserved through passaging, with low levels of replicative senescence. In vivo, SV-APC transplantation improved blood flow recovery and revascularization of ischemic limbs. Whole genome screening showed an association between DNA methylation at the promoter or gene body level and microvascular density and to a lesser extent with blood flow recovery. Expressional studies highlighted the implication of an angiogenic network centered on the vascular endothelial growth factor receptor as a predictor of microvascular outcomes. FLT-1 gene silencing in SV-APCs remarkably reduced their ability to form tubes in vitro and support tube formation by human umbilical vein endothelial cells, thus confirming the importance of this signaling in SV-APC angiogenic function. CONCLUSIONS: DNA methylation landscape illustrates different therapeutic activities of human APCs. Epigenetic screening may help identify determinants of therapeutic vasculogenesis in ischemic disease.

A journey from basic stem cell discovery to clinical application: the case of adventitial progenitor cells.

Ischemia is a leading cause of death in the western world. Regenerative medicine aims to improve healing of ischemic injury by complementing pharmacologic/interventional treatments. Navigating regenerative therapies from 'bench-to-bedside' is a multistep time-consuming process, balancing cell expansion, purity, safety and efficacy while complying with regulatory guidelines. Studies started in academic laboratories unused to long-term planning often fail because of poor strategy design, lack of contingency plans or funding. We provide a strategic insight into our translation of saphenous vein-derived adventitial progenitor cells into a clinical grade product to treat angina. We discuss discovery phases, introduction of standard operating procedures and upgrade to clinical standards. We also examine contractual aspects of transferring to GMP-accredited facilities for clinical production and unexpected hurdles.

Chronic exposure to laminar shear stress induces Kruppel-like factor 2 in glomerular endothelial cells and modulates interactions with co-cultured podocytes.

Laminar shear stress (LSS), induced by flowing blood, plays a key role in determining vascular health by modulating endothelial behaviour and vascular tone. In systemic endothelium many of the beneficial effects of chronic LSS are mediated through the transcription factor Kruppel-like factor 2 (KLF2), but little is known regarding the role of chronic LSS in the renal glomerulus. We demonstrate that exposure of glomerular endothelial cells to chronic (>24h) LSS of 10 dyn/cm(2) increases phosphorylation of extra-cellular signal-related kinase 5 (ERK5) and increases expression of KLF2, leading to increased expression of the downstream molecules endothelial nitric oxide synthase (eNOS), thrombomodulin, endothelin-1 and nitric oxide. However, the proportion of eNOS which was phosphorylated at serine 1117 and threonine 495 residues was decreased. We demonstrated dependence of these effects on the ERK5 pathway by using the inhibitor UO126. We found high levels of KLF2 expression in human glomeruli confirming the relevance of our in vitro observations and, as KLF2 is specifically induced by chronic LSS, suggesting the physiological importance of shear stress in the glomerulus. Conditioned medium from glomerular endothelial cells under chronic LSS decreased podocyte monolayer resistance and increased phosphorylation of vasodilator-stimulated phosphoprotein. The latter effect was more pronounced using a novel insert-based direct co-culture system in which endothelial cells were exposed to chronic LSS. These data provide the first direct evidence of glomerular endothelial cell to podocyte cross-talk.

An in vitro model of the glomerular capillary wall using electrospun collagen nanofibres in a bioartificial composite basement membrane.

The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease.

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase.

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.

MMP-9 and -12 cause N-cadherin shedding and thereby beta-catenin signalling and vascular smooth muscle cell proliferation.

AIMS: Vascular smooth muscle cell (VSMC) proliferation contributes to intimal thickening in restenosis and atherosclerosis. Previously, we demonstrated that matrix-degrading metalloproteinase (MMP)-dependent shedding of the extracellular portion of N-cadherin increased VSMC proliferation via elevation of beta-catenin signalling and cyclin D1 expression. In this study, we aimed to determine whether MMP-2, -9, -12, or -14 regulates VSMC proliferation via N-cadherin shedding. METHODS AND RESULTS: N-cadherin shedding was significantly impaired in proliferating mouse aortic VSMCs deficient in MMP-9 (MMP-9(-/-)) and MMP-12 (MMP-12(-/-)) compared with wild-type controls (1.1 +/- 0.7- and 1.0 +/- 0.1- vs. 2.0 +/- 0.2-fold). Furthermore, proliferating VSMCs subjected to MMP-9 or -12 siRNA knockdown or deficient in MMP-9 or -12 showed significantly increased cellular levels of N-cadherin compared with controls (1.7 +/- 0.2-, 2.7 +/- 0.6-, and 3.5 +/- 1.6-, 1.7 +/- 0.2-fold, respectively). Incubation of VSMCs with active MMP-9 or -12 independently increased N-cadherin cleavage. Additionally, beta-catenin signalling was significantly reduced by 52 +/- 17 and 81 +/- 12% in MMP-9(-/-) and -12(-/-) proliferating VSMCs, respectively, and this was corroborated by siRNA knockdown of MMP-9 and -12. Decreased beta-catenin signalling coincided with significantly reduced proliferation and cyclin D1 protein levels in MMP-9(-/-) and -12(-/-) cells. Little or no additive effect was observed with combined modulation of MMP-9 and -12 in all experiments. In contrast, N-cadherin shedding and VSMC proliferation were not modulated by MMP-2 and -14. CONCLUSION: In conclusion, we propose that MMP-9 and -12 promote intimal thickening by independent cleavage of N-cadherin, which elevates VSMC proliferation via beta-catenin signalling.

Vascular endothelial growth factor-C, a potential paracrine regulator of glomerular permeability, increases glomerular endothelial cell monolayer integrity and intracellular calcium.

We have previously reported expression of vascular endothelial growth factor (VEGF)-A and -C in glomerular podocytes and actions of VEGF-A on glomerular endothelial cells (GEnC) that express VEGF receptor-2 (VEGFR-2). Here we define VEGFR-3 expression in GEnC and investigate the effects of the ligand VEGF-C. Renal cortex and cultured GEnC were examined by microscopy, and both cell and glomerular lysates were assessed by Western blotting. VEGF-C effects on trans-endothelial electrical resistance and albumin flux across GEnC monolayers were measured. The effects of VEGF-C156S, a VEGFR-3-specific agonist, and VEGF-A were also studied. VEGF-C effects on intracellular calcium ([Ca2+]i) were measured using a fluorescence technique, receptor phosphorylation was examined by immunoprecipitation assays, and phosphorylation of myosin light chain-2 and VE-cadherin was assessed by blotting with phospho-specific antibodies. GEnC expressed VEGFR-3 in tissue sections and culture, and VEGF-C increased trans-endothelial electrical resistance in a dose-dependent manner with a maximal effect at 120 minutes of 6.8 Omega whereas VEGF-C156S had no effect. VEGF-C reduced labeled albumin flux by 32.8%. VEGF-C and VEGF-A increased [Ca2+]i by 15% and 39%, respectively. VEGF-C phosphorylated VEGFR-2 but not VEGFR-3, myosin light chain-2, or VE-cadherin. VEGF-C increased GEnC monolayer integrity and increased [Ca2+]i, which may be related to VEGF-C-S particular receptor binding and phosphorylation induction characteristics. These observations suggest that podocytes direct GEnC behavior through both VEGF-C and VEGF-A.

Regulation of smooth muscle cell proliferation by beta-catenin/T-cell factor signaling involves modulation of cyclin D1 and p21 expression.

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of beta-catenin. In this study we demonstrate directly that growth factors stimulate beta-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether beta-catenin/TCF signaling regulates VSMC proliferation via modulation of the beta-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited beta-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of beta-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by beta-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting beta-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and beta-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 beta-catenin/TCF responsive genes.

N-cadherin-dependent cell-cell contacts promote human saphenous vein smooth muscle cell survival.

OBJECTIVE: Vascular smooth muscle cell (VSMC) apoptosis is thought to contribute to atherosclerotic plaque instability. Cadherin mediates calcium-dependent homophilic cell-cell contact. We studied the role of N-cadherin in VSMC apoptosis. METHODS AND RESULTS: Human saphenous vein VSMCs were grown in agarose-coated wells to allow cadherin-mediated aggregate formation. Cell death and apoptosis were determined after disruption of cadherins using several approaches (n> or =3 per approach). Calcium removal from culture medium or addition of nonspecific cadherin antagonist peptides significantly decreased aggregate formation and increased cell death by apoptosis (34+/-6% versus 75+/-1% and 19+/-1% versus 40+/-5%, respectively; P<0.05). Specific inhibition of N-cadherin using antagonists and neutralizing antibodies similarly increased apoptosis. Supporting this, overexpression of full-length N-cadherin significantly reduced VSMC apoptosis from 44+/-10% to 20+/-3% (P<0.05), whereas abolishing N-cadherin expression by overexpression of a dominant-negative N-cadherin significantly, even in the presence of cell-matrix contacts, increased apoptosis from 9+/-2% to 50+/-1% (P<0.05). Interestingly, cell-cell contacts provided a similar degree of protection from apoptosis to cell-matrix contacts. Finally, N-cadherin-mediated cell-cell contacts initiated anti-apoptotic signaling by increasing Akt and Bad phosphorylation. CONCLUSIONS: Our results indicate that VSMC survival is dependent on N-cadherin-mediated cell-cell contacts, which could be important in the context of plaque instability.

R-cadherin:beta-catenin complex and its association with vascular smooth muscle cell proliferation.

OBJECTIVE: Vascular smooth muscle cell (VSMC) proliferation is an important component of atherosclerosis, restenosis after angioplasty and stent placement, and vein graft failure. Outside-in signaling from the cadherin:beta-catenin complex can increase transcription of the cell-cycle gene cyclin D1; however, its role in VSMC proliferation has only recently been considered. METHODS AND RESULTS: We examined the involvement of R-cadherin and beta-catenin in VSMC proliferation in balloon-injured carotid arteries in vivo and aortic rings in vitro. The number of medial VSMCs positive for R-cadherin was significantly reduced by 32%+/-5%, 52%+/-10%, and 23%+/-2% at 0.25, 24, and 48 hours after injury in vivo, respectively. These changes in cadherin expression coincided with the detection of nuclear beta-catenin and elevated cyclin D1 expression. Furthermore, loss of R-cadherin expression was associated with medial VSMC proliferation. Inhibition of classical cadherin function with a HAV peptide and R-cadherin neutralizing antibodies significantly increased proliferation by 4.3+/-1.0-fold and 4.1+/-0.98-fold, and increased the number of cells with beta-catenin in the nucleus and expressing cyclin D1 in aortic rings. CONCLUSIONS: These results suggest that R-cadherin expression and beta-catenin signaling may be associated with increased cyclin D1 expression and VSMC proliferation and may therefore play an important role in vascular disease.