Pericyte Precursors Research Articles

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Abstract 2880: Radiation induces myofibroblast differentiation of pericytes

Abstract Radiation not only kills cancer cells directly through DNA damage, but also indirectly by inducing vascular changes. Endothelial cells (ECs) in small blood vessels and capillaries connect with pericytes, a type of mural cells which are important for vascular stability and structure. Effects of radiation on tumor vasculature are critical for the therapeutic efficacy, however, its impacts on pericytes remain largely unknown. In this study, we focused on the effect of radiation on pericytes and sought to elucidate the mechanism by which radiation affects pericytes. We show 20Gy for C3H10T1/2 (10T1/2) mouse embryonic mesenchymal pericyte precursor cells and 10Gy for human pericytes from placenta (hPC-PL) cells induces pericyte differentiation and activation of adhesion molecules. Radiation enhanced lymphocyte adhesion and decreased permeability in co-culture of ECs and pericytes primarily through endothelial cells, not via pericytes. Furthermore, radiation promoted transdifferentiation of pericytes into myofibroblasts. Supernatant from irradiated cells in combination with a TGFβ inhibitor showed that TGFβ signaling is involved in pericyte differentiation by radiation but not all pericyte marker genes are regulated by TGFβ. Therefore, we performed phospho-kinase array analysis and revealed Akt signaling pathway activation 10 minutes following radiation, which was verified by the phosphorylation of GSK3β and PRAS40. Along with this, generation of intracellular reactive oxygen species (ROS) occurs as early as 5 min following radiation. Inhibitors of Akt and ROS suppress the expression of myofibroblast markers increased by radiation. To investigate whether radiation induces pericyte differentiation into myofibroblast in tumors, we employed NG2DsRedBAC (NG2DsRed) transgenic mice where DsRed is expressed under the control of NG2 promoter to trace pericytes. The endogenous expression of DsRed was detected in pericytes in the tumors implanted to NG2DsRed mice. Flow cytometry analysis of tumors harvested 7 days after radiation shows radiation increased vimentin+ cells and decreased DsRed+ cells, which indicates radiation induced pericyte differentiation into myofibroblast. In conclusion, radiation promotes pericyte maturation and transdifferentiation into myofibroblast via Akt signaling. Further study using Akt inhibitor with radiation to tumor-bearing N2DsRed mice will be warranted. Citation Format: Tomoko Yamazaki, Kelley R. Jordan, Marka R. Crittenden, Michael J. Gough, Kristina H. Young. Radiation induces myofibroblast differentiation of pericytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2880.

Mesenchymal Stem Cells Potentiate the Vasculogenic Capacity of Endothelial Colony-Forming Cells under Hyperglycemic Conditions.

Many studies have demonstrated a reduced number and vasculogenic capacity of endothelial colony-forming cells (ECFCs) in diabetic patients. However, whether the vasculogenic capacity of ECFCs is recovered or not when combined with pericyte precursors, mesenchymal stem cells (MSCs), under hyperglycemic conditions has not been studied. Thus, we investigated the role of MSCs in ECFC-mediated vascular formation under high-glucose conditions. The ECFCs and MSCs were treated with normal glucose (5 mM; NG) or high glucose (30 mM; HG) for 7 days. The cell viability, proliferation, migration, and tube formation of ECFCs were reduced in HG compared to NG. Interestingly, the ECFC+MSC combination after HG treatment formed tubular structures similar to NG-treated ECFCs+MSCs. An in vivo study using a diabetic mouse model revealed that the number of perfused vessels formed by HG-treated ECFCs+MSCs in diabetic mice was comparable with that of NG-treated ECFCs+MSCs in normal mice. Electron microscopy revealed that the ECFCs+MSCs formed pericyte-covered perfused blood vessels, while the ECFCs alone did not form perfused vessels when injected into the mice. Taken together, MSCs potentiate the vasculogenic capacity of ECFCs under hyperglycemic conditions, suggesting that the combined delivery of ECFCs+MSCs can be a promising strategy to build a functional microvascular network to repair vascular defects in diabetic ischemic regions.

Decoding myofibroblast origins in human kidney fibrosis.

Kidney fibrosis is the hallmark of chronic kidney disease progression, however, currently no antifibrotic therapies exist. This is largely because the origin, functional heterogeneity and regulation of scar-forming cells during human kidney fibrosis remains poorly understood. Here, using single cell RNA-seq, we profiled the transcriptomes of proximal tubule and non-proximal tubule cells in healthy and fibrotic human kidneys to map the entire human kidney in an unbiased approach. This enabled mapping of all matrix-producing cells at high resolution, revealing distinct subpopulations of pericytes and fibroblasts as the major cellular sources of scar forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single cell RNA-seq and ATAC-seq experiments in mice, and spatial transcriptomics in human kidney fibrosis to functionally interrogate these findings, shedding new light on the origin, heterogeneity and differentiation of human kidney myofibroblasts and their fibroblast and pericyte precursors at unprecedented resolution. Finally, we used this strategy to facilitate target discovery, identifying Nkd2 as a myofibroblast-specific target in human kidney fibrosis.

Dual function of perivascular fibroblasts in vascular stabilization in zebrafish.

Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular fibroblasts markedly reduces collagen deposition around endothelial cells, resulting in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular stabilization where they establish the ECM around nascent vessels and function as pericyte progenitors.

New Pericytes in the Adult Brain Originate from a Previously Unrecognized Pericyte Precursor

Pericytes are essential for maintaining the stability of CNS blood-vessels and the blood-brain- barrier. They are closely associated with endothelia and can be identified by a set of markers. New pericytes are required during neo-angiogenesis but how pericytes are generated in the adult brain is unknown. Here, we investigated neoplastic vascularization of the adult brain in a transgenic lineage-tracing model. We observed that mature pericytes largely originate from a newly identified precursor celltype, which is distinct from established brain cell-populations as evidenced by single-cell RNAseq studies. These precursors are characterized as pericyte marker-negative cells of non-hematopoietic origin, which are distant from blood-vessels and highly proliferative. Pericyte precursors emerge also in developmental angiogenesis and lineage ablation experiments in brain tumor models showed that they are necessary to generate new, mature pericytes supporting tumor expansion. Overall, pericyte precursors control vascular plasticity and provide an excellent therapeutic target for brain tumors.

A Novel Population of Local Pericyte Precursor Cells in Tumor Stroma that Require Notch Signaling for Differentiation

A Novel Population of Local Pericyte Precursor Cells in Tumor Stroma that Require Notch Signaling for Differentiation

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1+ precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146+/CD45−/CD31−/lo pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1hi/CD146−/CD45−/CD31−. The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1hi/CD146−/CD45−/CD31− cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1hi/CD146−/CD45−/CD31− cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1−/lo/CD146−/CD45−/CD31− cells. Our results suggest that a local Sca1hi/CD146−/CD45−/CD31− pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes.

Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)–sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion of MMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis.

A Novel Role for Mesenchymal Stem/Progenitor Cells As Hypoxia Sensors During Initiation of Neo-Vasculogenesis in Vivo

Abstract 613 Background:Hypoxia is a major stimulus of neo-vasculogenesis. Under hypoxic conditions endothelial colony-forming progenitor cells (ECFCs) arrange tubular structures, which can connect to the pre-existing vasculature forming functional perfused vessels. The current view is that mesenchymal stem and progenitor cells (MSPCs) or their pericyte progeny are recruited subsequently to stabilize vessels. So far, clinical applications of endothelial progenitors to restore tissue oxygenation after ischemia, cardiovascular disease or stroke largely failed to meet medical needs. Based on previous work demonstrating patent vessel formation after MSPC/ECFC co-transplantation in vivo (Blood 2009), we hypothesized that MSPCs have a decisive role in the vasculogenic response to hypoxia. Here we show for the first time that ECFCs in hypoxic conditions in vivo strictly require the presence of functional MSPCs not only to stabilize but primarily to initiate neo-vasculogenesis by a hypoxia-inducible transcription factor (HIF)-dependent mechanism. Methods:Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. Progenitor cell phenotype, long-term proliferation, HIF stabilization, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen (5% O2) and standard ambient air culture conditions (20% O2). ECFC and MSPC crosstalk in vivo was studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelet lysate gel). Cell type-specific chemical and genetic inhibition of HIF (YC-1, shRNA) was used to delineate the role of hypoxia sensing in MSPCs and ECFCs, respectively, during vasculogenesis in vivo. To determine if downstream target proteins of HIF-1α could substitute for MSPC presence during vasculogenesis, selected growth factors and cytokines were tested. Results:Progenitor proliferation and function in vitro were reduced with declining oxygen levels. ECFCs stabilized hypoxia-inducible factor-1α (HIF-1α) only at 1% O2, while MSPCs stabilized HIF-1α already at 5% O2. In an NSG mouse model, ECFCs transplanted into a hypoxic environment did not stabilize HIF-1α, while transplanted sole MSPCs or MSPCs in co-transplants showed strong nuclear HIF-1α stabilization 1 day after transplantation preceding any vessel formation or perfusion. In the absence of MSPCs, the majority of ECFCs underwent apoptosis within 24h in vivo. Inhibition of HIF-1α stabilization in MSPCs but not in ECFCs significantly abrogated vessel formation in vivo. Blocking the prominent HIF-1α down-stream target vascular endothelial growth factor (VEGF) resulted in the expected inhibition of neo-vasculogenesis. Interestingly, substitution of VEGF alone could not restore vessel formation, neither when injected together with sole ECFCs nor in a model where ECFCs were co-transplanted with HIF-depleted MSPCs. Substitution of a complex mixture of platelet-derived factors in vivo partly restored the vasculogenic function of HIF-depleted MSPCs. Conclusions:MSPCs react to a low oxygen environment by stabilizing HIF-1α earlier and more sensitively than ECFCs. MSPCs promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis in the initial phase of vasculogenesis by a HIF-dependent trophic mechanism. Surprisingly, therapeutic vasculogenesis can occur independently of endothelial HIF stabilization. These results argue in favor of MSPC/ECFC co-transplantation as a promising strategy for vascular regenerative therapy. The observation that VEGF alone could not compensate for the vasculogenic competence of pericyte precursors in vivo underlines the complexity of the hypoxia-induced cytokine network. The fact that hypoxia sensing in MSPCs but not in ECFCs is crucial to initiate vascular regeneration supports a shift of focus from endothelial cells to perivascular mesenchymal cells as a therapeutic target in anti-angiogenic therapy. Disclosures:No relevant conflicts of interest to declare.

Potentiation of Platelet-Derived Growth Factor Receptor-β Signaling Mediated by Integrin-Associated MFG-E8

Pericytes/pericyte precursors produce milk fat globule-associated protein with epidermal growth factor and factor VIII-like domains (MFG-E8) in vivo, and this α(v) integrin ligand enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice. Inhibition of MFG-E8 production or function attenuates platelet-derived growth factor-BB (PDGF-BB)-induced migration of pericyte/pericyte precursor-like 10T1/2 cells in vitro. Herein, we describe mechanisms by which MFG-E8 modulates PDGF-BB:PDGF receptor β (PDGFRβ) signaling in 10T1/2 cells. Small interfering RNA depletion of MFG-E8 from 10T1/2 cells or antibody inhibition of MFG-E8 action enhanced PDGF-BB-dependent degradation of PDGFRβ and attenuated signaling. Coimmunoprecipitation revealed transient association of MFG-E8 with PDGFRβ in PDGF-BB-treated 10T1/2 cells and reduced PDGFRβ-focal adhesion kinase association in MFG-E8-depleted cells. Confocal microscopy demonstrated that MFG-E8 binding to 10T1/2 cells was RGD motif and α(v) dependent but PDGF-BB treatment independent, whereas colocalization of MFG-E8 with PDGFRβ was enhanced by PDGF-BB. Ubiquitination of PDGFRβ was also increased in MFG-E8 small interfering RNA-transfected cells. Integrin α(v)-bound MFG-E8 associates with PDGFRβ and focal adhesion kinase after PDGF-BB treatment, results in cell surface retention of PDGFRβ, delays receptor degradation, potentiates downstream signaling, and enhances migration of 10T1/2 cells. MFG-E8 may promote angiogenesis, in part, via cell autonomous actions on pericytes or pericyte precursors that result in enhanced PDGF-BB:PDGFRβ signaling mediated via integrin-growth factor receptor cross-talk.

The role of pericytes in angiogenesis

Pericytes are branched cells embedded within the basement membrane of capillaries and post-capillary venules. They provide an incomplete investment to endothelial cells, thus reinforcing vascular structure and regulating microvascular blood flow. Pericytes exert an important role on endothelial cell proliferation, migration and stabilization. Endothelial cells, in turn, stimulate expansion and activation of the pericyte precursor cell population. The balance between the number of endothelial cells and pericytes is highly controlled by a series of signaling pathway mechanisms operating in an autocrine and/or paracrine manner. In this review, we will first examine the molecular aspects of the pericyte activating factors secreted by endothelial cells, such as platelet derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and angiopoietins (Angs), as well as signaling pathways involving Notch and ephrins. We will then consider the complex and multivarious contribution of pericytes to the different aspects of angiogenesis with particular emphasis on the potential role of these cells as targets in tumor therapy.

Covalently immobilized platelet-derived growth factor-BB promotes angiogenesis in biomimetic poly(ethylene glycol) hydrogels

The field of tissue engineering is severely limited by a lack of microvascularization in tissue engineered constructs. Biomimetic poly(ethylene glycol) hydrogels containing covalently immobilized platelet-derived growth factor BB (PDGF-BB) were developed to promote angiogenesis. Poly(ethylene glycol) hydrogels resist protein absorption and subsequent non-specific cell adhesion, thus providing a “blank slate”, which can be modified through the incorporation of cell adhesive ligands and growth factors. PDGF-BB is a key angiogenic protein able to support neovessel stabilization by inducing functional anastomoses and recruiting pericytes. Due to the widespread effects of PDGF in the body and a half-life of only 30 min in circulating blood, immobilization of PDGF-BB may be necessary. In this work bioactive, covalently immobilized PDGF-BB was shown to induce tubulogenesis on two-dimensional modified surfaces, migration in three-dimensional (3D) degradable hydrogels and angiogenesis in a mouse cornea micropocket angiogenesis assay. Covalently immobilized PDGF-BB was also used in combination with covalently immobilized fibroblast growth factor-2, which led to significantly increased endothelial cell migration in 3D degradable hydrogels compared with the presentation of each factor alone. When a co-culture of endothelial cells and mouse pericyte precursor 10T1/2 cells was seeded onto modified surfaces tubule formation was independent of surface modifications with covalently immobilized growth factors. Furthermore, the combination of soluble PDGF-BB and immobilized PDGF-BB induced a more robust vascular response compared with soluble PDGF-BB alone when implanted into an in vivo mouse cornea micropocket angiogenesis assay. Based on these results, we believe bioactive hydrogels can be tailored to improve the formation of functional microvasculature for tissue engineering.

STRO-1 + mesenchymal precursor cells located in synovial surface projections of patients with osteoarthritis

To investigate the presence of mesenchymal precursor cells (MPCs) in synovial surface projections of patients with osteoarthritis (OA), to characterize their phenotype and to show their localization. Progenitor cells in synovial surface projections were identified by immunohistochemistry, morphometric analysis and confocal laser scanning microscopy using the following phenotypic markers: STRO-1, CD34, and alpha smooth muscle actin (alpha-SMA). In the synovial tissue of all 21 patients with OA MPCs were detected. Immunohistochemistry and subsequent morphometric analysis showed that approximately twice as many STRO-1+ cells/mm2 were observed in synovial tissue of patients with OA as compared to healthy organ donors and that number of STRO-1+ cells/mm2 correlated with total cell number/mm2. Interestingly, in the synovial tissue of patients with OA, twice as many STRO-1+ cells/mm2 were found in synovial surface projections as compared to the sublining area without villi. Using confocal laser scanning microscopy two populations of STRO-1+ MPCs could be detected in synovial surface projections. Single STRO-1+ cells that co-expressed alpha-SMA resemble a population of pericyte precursors required to stabilize the immature vasculature. The second STRO-1+ cell population that was found lacked alpha-SMA but co-expressed CD34 on their surface with low intensity. Here we can show that in the synovial tissue of patients with OA twice as many STRO-1+ MPCs can be found in synovial surface projections as compared to the sublining area. These cells are preferentially located at the basis and in the protruding end of the synovial surface projection.

Contribution of Bone Marrow–Derived Pericyte Precursor Cells to Corneal Vasculogenesis

Bone-marrow (BM)-derived hematopoietic precursor cells are thought to participate in the growth of blood vessels during postnatal vasculogenesis. In this investigation, multichannel laser scanning confocal microscopy and quantitative image analysis were used to study the fate of BM-derived hematopoietic precursor cells in corneal neovascularization. A BM-reconstituted mouse model was used in which the BM from enhanced green fluorescent protein (GFP)-positive mice was transplanted into C57BL/6 mice. Basic fibroblast growth factor (bFGF) was used to induce corneal neovascularization in mice. The vasculogenic potential of adult BM-derived cells and their progeny were tested in this in vivo model. Seventy-two histologic sections selected by systematic random sampling from four mice were immunostained and imaged with a confocal microscope and analyzed with image-analysis software. BM-derived endothelial cells did not contribute to bFGF-induced neovascularization in the cornea. BM-derived periendothelial vascular mural cells (pericytes) were detected at sites of neovascularization, whereas endothelial cells of blood vessels originated from preexisting blood vessels in limbal capillaries. Fifty three percent of all neovascular pericytes originated from BM, and 47% of them originated from preexisting corneoscleral limbus capillaries. Ninety-six percent and 92% of BM-derived pericytes also expressed CD45 and CD11b, respectively, suggesting their hematopoietic origin from the BM. Pericytes of new corneal vessels have a dual source: BM and preexisting limbal capillaries. These findings establish BM as a significant effector organ in corneal disorders associated with neovascularization.

Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors

Bone marrow-derived cells contribute to tumor angiogenesis. Here, we demonstrate that monocytes expressing the Tie2 receptor (Tie2-expressing monocytes [TEMs]) (1) are a distinct hematopoietic lineage of proangiogenic cells, (2) are selectively recruited to spontaneous and orthotopic tumors, (3) promote angiogenesis in a paracrine manner, and (4) account for most of the proangiogenic activity of myeloid cells in tumors. Remarkably, TEM knockout completely prevented human glioma neovascularization in the mouse brain and induced substantial tumor regression. Besides TEMs and endothelial cells (ECs), Tie2 expression distinguished a rare population of tumor stroma-derived mesenchymal progenitors representing a primary source of tumor pericytes. Therefore, Tie2 expression characterizes three distinct cell types required for tumor neovascularization: ECs, proangiogenic cells of hematopoietic origin, and pericyte precursors of mesenchymal origin.

Endothelial–Mesenchymal Interactions In Vitro Reveal Molecular Mechanisms of Smooth Muscle/Pericyte Differentiation

Cell-cell interactions are central to vascular development. We have developed an in vitro system in which endothelial cells (EC) are co-cultured with 10T1/2 cells as smooth muscle cell (SMC)/pericyte precursors. 10T1/2 cells, in contact with EC, differentiate to SMC in a process mediated, at least in part, by a transforming growth factor-beta (TGF-beta)-mediated event. Co-culture with EC or TGF-beta treatment induced expression of SM22alpha, with co-culture inducing a significantly greater response. To dissect the molecular mechanisms of SMC/pericyte differentiation, reporter constructs containing the promoter for SM22alpha, a SMC-specific gene, were stably transfected into 10T1/2 cells and response to EC-co-culture and TGFbeta were compared. Co-culture with EC or TGFbeta treatment stimulated activity of a 441-bp SM22-alpha promoter to about the same extent, whereas co-culture induced the activity of a 3.7-kb promoter to about twice that of TGBbeta. Neutralization of TGFbeta in EC-10T1/2 co-cultures partially reduced the 3.7-kb SM22alpha promoter activity in 10T1/2 cells. Previously unidentified CArG and TCE elements near the 5' end of the promoter are responsible for full promoter activity. EC-mesenchymal contact appears to be required for full promoter activity of the SM22alpha gene in 10T1/2 and requires upstream CArG and TCE elements. The 3.7-kb SM22alpha promoter can direct expression of lacZ in vivo to SMC of the large vessels and the smaller intersomitic vessels. We have identified the expression of SM22alpha in pericytes of the retinal microvasculature in developing and remodeling vessels.

Endothelial–Mesenchymal Interactions In Vitro Reveal Molecular Mechanisms of Smooth Muscle/Pericyte Differentiation

Cell–cell interactions are central to vascular development. We have developed an in vitro system in which endothelial cells (EC) are co-cultured with 10T1/2 cells as smooth muscle cell (SMC)/pericyte precursors. 10T1/2 cells, in contact with EC, differentiate to SMC in a process mediated, at least in part, by a transforming growth factor-β (TGF-β)-mediated event. Co-culture with EC or TGF-β treatment induced expression of SM22α, with co-culture inducing a significantly greater response. To dissect the molecular mechanisms of SMC/pericyte differentiation, reporter constructs containing the promoter for SM22α, a SMC-specific gene, were stably transfected into 10T1/2 cells and response to EC-co-culture and TGFβ were compared. Co-culture with EC or TGFβ treatment stimulated activity of a 441-bp SM22-α promoter to about the same extent, whereas co-culture induced the activity of a 3.7-kb promoter to about twice that of TGBβ. Neutralization of TGFβ in EC-10T1/2 co-cultures partially reduced the 3.7-kb SM22α promoter activity in 10T1/2 cells. Previously unidentified CArG and TCE elements near the 5' end of the promoter are responsible for full promoter activity. EC–mesenchymal contact appears to be required for full promoter activity of the SM22α gene in 10T1/2 and requires upstream CArG and TCE elements. The 3.7-kb SM22α promoter can direct expression of lacZ in vivo to SMC of the large vessels and the smaller intersomitic vessels. We have identified the expression of SM22α in pericytes of the retinal microvasculature in developing and remodeling vessels.