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DCBLD2 is a neuropilin-like transmembrane protein that is up-regulated during arterial remodeling in humans, rats, and mice. Activation of PDGFR-β via PDGF triggers receptor phosphorylation and endocytosis. Subsequent activation of downstream signals leads to the stimulation of phenotypic conversion of VSMCs and arterial wall proliferation, which are common pathological changes in vascular remodeling diseases such as atherosclerosis, hypertension, and restenosis after angioplasty. In this study, we hypothesized that DCBLD2 regulates neointimal hyperplasia through the regulation of PDGFR-β endocytosis of vascular smooth muscle cells (VSMCs) through Caveolin-1 (Cav-1). Compared with wild-type (WT) mice or control littermate mice, the germline or VSMC conditional deletion of the Dcbld2 gene resulted in a significant increase in the thickness of the tunica media in the carotid artery ligation. To elucidate the underlying molecular mechanisms, VSMCs were isolated from the aorta of WT or Dcbld2 -/- mice and were stimulated with PDGF. Western blotting assays demonstrated that Dcbld2 deletion increased the PDGF signaling pathway. Biotin labeling test and membrane-cytosol separation test showed that after DCBLD2 was knocked down or knocked out, the level of PDGFR-β on the cell membrane was significantly reduced, while the amount of PDGFR-β in the cytoplasm increased. Co-immunoprecipitation experiments showed that after DCBLD2 gene knock-out, the binding of PDGFR-β and Cav-1 in the cytoplasm significantly increased. Double immunofluorescence staining showed that PDGFR-β accumulated Cav-1/lysosomes earlier than for control cells, which indicated that DCBLD2 gene knock-down or deletion accelerated the endocytosis of PDGF-induced PDGFR-β in VSMCs. In order to confirm that DCBLD2 affects the relationship between Cav-1 and PDGFR-β, proteins extracted from VSMCs cultured in vitro were derived from WT and Dcbld2 -/- mice, whereas co-immunoprecipitation suggested that the combination of DCBLD2 and Cav-1 reduced the bond between Cav-1 and PDGFR-β, and DCBLD2 knock-out was able to enhance the interaction between Cav-1 and PDGFR-β. Therefore, the current results suggest that DCBLD2 may inhibit the caveolae-dependent endocytosis of PDGFR-β by anchoring the receptor on the cell membrane. Based on its ability to regulate the activity of PDGFR-β, DCBLD2 may be a novel therapeutic target for the treatment of cardiovascular diseases., (© 2022 Federation of American Societies for Experimental Biology.)

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration, and remodeling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular, and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. We used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single-cell RNA sequencing to redefine the endometrial mesenchyme. In contrast to previous reports we show that CD146 is expressed in both PDGFRβ + perivascular cells and CD31 + endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express the high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, and Abcc9). scRNA-seq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34 + but were distinct in their expression of Ngfr/Spon2/Angptl7 (F1), Cxcl14/Smoc2/Rgs2 (F2), and Clec3b/Col14a1/Mmp3 (F3), with potential functions in the regulation of immune responses, response to wounding, and organization of extracellular matrix, respectively. Immunohistochemistry was used to investigate the spatial distribution of these populations revealing F1/NGFR + cells in most abundance beside epithelial cells. We provide the first definitive analysis of mesenchymal cells in the adult mouse endometrium identifying five subpopulations providing a platform for comparisons between mesenchymal cells in endometrium and other adult tissues which are prone to fibrosis., (© 2020 Federation of American Societies for Experimental Biology.)

Pericytes promote vessel stability and their dysfunction causes pathologies due to blood vessel leakage. Previously, we reported that Olfactomedin-like 3 (Olfml3) is a matricellular protein with proangiogenic properties. Here, we explored the role of Olfml3 in a knockout mouse model engineered to suppress this protein. The mutant mice exhibited vascular defects in pericyte coverage, suggesting that pericytes influence blood vessel formation in an Olfml3-dependent manner. Olfml3-deficient mice exhibited abnormalities in the vasculature causing partial lethality of embryos and neonates. Reduced pericyte coverage was observed at embryonic day 12.5 and persisted throughout development, resulting in perinatal death of 35% of Olfml3-deficient mice. Cultured Olfml3-deficient pericytes exhibited aberrant motility and altered pericyte association to endothelial cells. Furthermore, the proliferative response of Olfml3 -/- pericytes upon PDGF-B stimulation was significantly diminished. Subsequent experiments revealed that intact PDGF-B signaling, mediated via Olfml3 binding, is required for pericyte proliferation and activation of downstream kinase pathways. Our findings suggest a model wherein pericyte recruitment to endothelial cells requires Olfml3 to provide early instructive cue and retain PDGF-B along newly formed vessels to achieve optimal angiogenesis., (© 2020 Federation of American Societies for Experimental Biology.)

Nearly 6 million Americans suffer from heart failure. Increased fibrosis contributes to functional decline of the heart that leads to heart failure. Previously, we identified a mechanosensitive protein, small proline-rich repeat 3 (SPRR3), in vascular smooth muscle cells of atheromas. In this study, we demonstrate SPRR3 expression in cardiac fibroblasts which is induced in activated fibroblasts following pressure-induced heart failure. Sprr3 deletion in mice showed preserved cardiac function and reduced interstitial fibrosis in vivo and reduced fibroblast proliferation and collagen expression in vitro. SPRR3 loss resulted in reduced activation of Akt, FAK, ERK, and p38 signaling pathways, which are coordinately regulated by integrins and growth factors. SPRR3 deletion did not impede integrin-associated functions including cell adhesion, migration, or contraction. SPRR3 loss resulted in reduced activation of PDGFRβ in fibroblasts. This was not due to the reduced PDGFRβ expression levels or decreased binding of the PDGF ligand to PDGFRβ. SPRR3 facilitated the association of integrin β1 with PDGFRβ and subsequently fibroblast proliferation, suggesting a role in PDGFRβ-Integrin synergy. We postulate that SPRR3 may function as a conduit for the coordinated activation of PDGFRβ by integrin β1, leading to augmentation of fibroblast proliferation and matrix synthesis downstream of biomechanical and growth factor signals., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Vascular smooth muscle cells (VSMCs) in the normal arterial media continually express contractile phenotypic markers which are reduced dramatically in response to injury. Tripartite motif-containing proteins are a family of scaffold proteins shown to regulate gene silencing, cell growth, and differentiation. We here investigated the biological role of tripartite motif-containing 28 (TRIM28) and tripartite motif-containing 27 (TRIM27) in VSMCs. We observed that siRNA-mediated knockdown of TRIM28 and TRIM27 inhibited platelet-derived growth factor (PDGF)-induced migration in human VSMCs. Both TRIM28 and TRIM27 can regulate serum response element activity and were required for maintaining the contractile gene expression in human VSMCs. At the same time, TRIM28 and TRIM27 knockdown reduced the expression of PDGF receptor-β (PDGFRβ) and the phosphorylation of its downstream signaling components. Immunoprecipitation showed that TRIM28 formed complexes with TRIM27 through its N-terminal RING-B boxes-Coiled-Coil domain. Furthermore, TRIM28 and TRIM27 were shown to be upregulated and mediate the VSMC contractile marker gene and PDGFRβ expression in differentiating human bone marrow mesenchymal stem cells. In conclusion, we identified that TRIM28 and TRIM27 cooperatively maintain the endogenous expression of PDGFRβ and contractile phenotype of human VSMCs., (© 2020 Federation of American Societies for Experimental Biology.)

Intercellular adhesion through homotypic interaction between cadherins regulates multiple cellular processes including cytoskeletal organization, proliferation, and survival. In this paper, we provide evidence that cadherin-11 (CDH11) binds to and promotes cell proliferation both in vitro and in vivo in synergy with the platelet-derived growth factor receptor beta (PDGFRβ). Engagement of CDH11 increased the sensitivity of cells to PDGF-BB by 10- to 100-fold, resulting in rapid and sustained phosphorylation of AKT, ultimately promoting and cell proliferation and tissue regeneration. Indeed, wound healing experiments showed that healing was severely compromised in Cdh11 -/- mice, as evidenced by significantly decreased proliferation, AKT phosphorylation, and extracellular matrix (ECM) synthesis of dermal cells. Our results shed light into understanding how intercellular adhesion can promote cell proliferation and may have implications for tissue regeneration and cancer progression., (© 2020 Federation of American Societies for Experimental Biology.)

Poststroke recovery requires multiple repair mechanisms, including vascular remodeling and blood-brain barrier (BBB) restoration. Brain pericytes are essential for BBB repair and angiogenesis after stroke, but they also give rise to scar-forming platelet-derived growth factor receptor β (PDGFR-β)-expressing cells. However, many of the molecular mechanisms underlying this pericyte response after stroke still remain unknown. Regulator of G-protein signaling 5 (RGS5) has been associated with pericyte detachment from the vascular wall, but whether it regulates pericyte function and vascular stabilization in the chronic phase of stroke is not known. Using RGS5-knockout (KO) mice, we study how loss of RGS5 affects the pericyte response and vascular remodeling in a stroke model at 7 d after ischemia. Loss of RGS5 leads to a shift toward an increase in the number of perivascular pericytes and reduction in the density of parenchymal PDGFR-β-expressing cells associated with normalized PDGFR-β activation after stroke. The redistribution of pericytes resulted in higher pericyte coverage, increased vascular density, preservation of vessel lengths, and a significant reduction in vascular leakage in RGS5-KO mice compared with controls. Our study demonstrates RGS5 in pericytes as an important target to enhance vascular remodeling.-Roth, M., Gaceb, A., Enström, A., Padel, T., Genové, G., Özen, I., Paul, G. Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke.

Platelet-derived growth factor receptor β (PDGFRβ) dynamically changes after brain injury, possibly mediating the neuroprotective role of soluble homodimers of the platelet-derived growth factor β subunit (PDGF-BB) that is secreted by microcirculation cells. The aim of this study was to determine whether binding of PDGF-BB to astrocytic PDGFRβ enhanced crosstalk among the various components of the neurovascular unit, leading to synaptic recovery after subarachnoid hemorrhage (SAH). The soluble PDGF-BB from the cerebrospinal fluid (CSF) of patients with SAH was measured. The relationship between PDGF-BB treatment and astrocytic PDGFRβ signaling was further explored in vivo and in vitro in experimental SAH models. Compared with the levels in the control samples, the PDGF-BB protein levels in the CSF of patients with SAH were significantly increased. After the generation of experimental SAH, astrocyte activation markers were markedly induced by the binding of PDGF-BB to astrocytic PDGFRβ, accompanied by improved levels of synaptic recovery and cognitive function. Soluble PDGF-BB and astrocytic PDGFRβ signaling are essential for the neuroprotective effect in the hippocampus and the coculture system in vitro after SAH that otherwise leads to cognitive dysfunction and neuronal damage.-Zhou, X., Wu, Q., Lu, Y., Zhang, X., Lv, S., Shao, J., Zhou, Y., Chen, J., Hou, L., Huang, C., Zhang, X. Crosstalk between soluble PDGF-BB and PDGFRβ promotes astrocytic activation and synaptic recovery in the hippocampus after subarachnoid hemorrhage.

The transformation of fibroblasts to myofibroblasts plays a major role in fibrogenic responses during dermal wound healing. We show a contribution of calpain systems (intracellular regulatory protease systems) in vascular endothelial cells (ECs) to myofibroblast differentiation in wound sites. Dermal wound healing experiments in mice found that calpastatin (an endogenous inhibitor of calpains) is enriched in preexisting vessels but not in newly formed capillaries. Transgenic overexpression of calpastatin in ECs delayed wound healing in mice as well as reducing the keratinocyte layer, extracellular matrix deposition, and myofibroblast accumulation in wound sites. EC and leukocyte markers, however, remain unchanged. Calpastatin overexpression reduced the expression of genes encoding platelet-derived growth factor-B and PDGF receptor-β (PDGFR-β). Topical application of platelet-derived growth factor-BB-containing ointment to wounds accelerated healing in control mice, but calpastatin overexpression prevented this acceleration. In cultured human dermal fibroblasts, α-smooth muscle actin and PDGFR-β were up-regulated by coculturing with ECs, but this action was inhibited by suppression of EC calpain activity. EC-driven transformation of mouse dermal fibroblasts was also suppressed by calpastatin overexpression in ECs. These results suggest that endothelial calpain systems influence PDGFR-β signaling in fibroblasts, EC-driven myofibroblast differentiation, and subsequent fibrogenic responses in wounds.-Miyazaki, T., Haraguchi, S., Kim-Kaneyama, J.-R., Miyazaki, A. Endothelial calpain systems orchestrate myofibroblast differentiation during wound healing.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-β, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca 2+ /calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Renal fibrosis leads to end-stage renal disease demanding renal replacement therapy because no adequate treatment exists. IFN-γ is an antifibrotic cytokine that may attenuate renal fibrosis. Systemically administered IFN-γ causes side effects that may be prevented by specific drug targeting. Interstitial myofibroblasts are the effector cells in renal fibrogenesis. Here, we tested the hypothesis that cell-specific delivery of IFN-γ to platelet-derived growth factor receptor β (PDGFRβ)-expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN-γ conjugated to PDGFRβ-recognizing peptide [(PPB)-polyethylene glycol (PEG)-IFN-γ] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN-γ. PDGFRβ expression was >3-fold increased (P < 0.05) in mouse fibrotic UUO kidneys and colocalized with α-smooth muscle actin-positive (SMA(+)) myofibroblasts. In vitro, PPB-PEG-IFN-γ significantly inhibited col1a1, col1a2, and α-SMA mRNA expression in TGF-β-activated NIH3T3 fibroblasts (P < 0.05). In vivo, PPB-PEG-IFN-γ specifically accumulated in PDGFRβ-positive myofibroblasts. PPB-PEG-IFN-γ treatment significantly reduced renal collagen I, fibronectin, and α-SMA mRNA and protein expression. Compared with vehicle treatment, PPB-PEG-IFN-γ preserved tubular morphology, reduced interstitial T-cell infiltration, and attenuated lymphangiogenesis (all P < 0.05) without affecting peritubular capillary density. PPB-PEG-IFN-γ reduced IFN-γ-related side effects as manifested by reduced major histocompatibility complex class II expression in brain tissue (P < 0.05 vs. free IFN-γ). Our findings demonstrate that specific targeting of IFN-γ to PDGFRβ-expressing myofibroblasts attenuates renal fibrosis and reduces systemic adverse effects., (© FASEB.)

Duchenne-Meryon muscular dystrophy (DMD) is the most common and lethal genetic muscle disease. Ameliorating muscle necrosis, inflammation, and fibrosis represents an important therapeutic approach for DMD. Imatinib, an antineoplastic agent, demonstrated antiinflammatory and antifibrotic effects in liver, kidney, lung, and skin of various animal models. This study tested antiinflammatory and antifibrotic effects of imatinib in mdx mice, a DMD mouse model. We treated mdx mice with intraperitoneal injections of imatinib at the peak of limb muscle inflammation and the onset of diaphragm fibrosis. Controls received PBS vehicle or were left untreated. Muscle necrosis, inflammation, fibrosis, and function were evaluated by measuring serum CK activity, endomysial CD45 immunoreactive inflammation area, endomysial collagen III deposition, and hind limb grip strength. Phosphorylation of the tyrosine kinase targets of imatinib was assessed by Western blot in diaphragm tissue and in primary cultures of peritoneal macrophages and skeletal muscle fibroblasts. Imatinib markedly reduced muscle necrosis, inflammation, and fibrosis, and significantly improved hind limb grip strength in mdx mice. Reduced clinical disease was accompanied by inhibition of c-abl and PDGFR phosphorylation and suppression of TNF-alpha and IL-1beta expression. Imatinib therapy for DMD may hold promise for ameliorating muscle necrosis, inflammation, and fibrosis by inhibiting c-abl and PDGFR signaling pathways and downstream inflammatory cytokine and fibrotic gene expression.

Preclinical and clinical evaluations of individual proangiogenic/arteriogenic factors for the treatment of ischemic myocardium and skeletal muscle have produced unfulfilled promises. The establishment of functional and stable arterial vascular networks may require combinations of different angiogenic and arteriogenic factors. Using in vivo angiogenesis and ischemic hind-limb animal models, we have compared the angiogenic and therapeutic activities of fibroblast growth factor 2 (FGF-2) in combinations with PDGF-AA and PDGF-AB, two members of the platelet-derived growth factor (PDGF) family, with distinct receptor binding patterns. We show that both PDGF-AA/FGF-2 and PDGF-AB/FGF-2 in combinations synergistically induce angiogenesis in the mouse cornea. FGF-2 up-regulates PDGFR-alpha and -beta expression levels in the newly formed blood vessels. Interestingly, PDGF-AB/FGF-2, but not PDGF-AA/FGF-2, is able to stabilize the newly formed vasculature by recruiting pericytes, and an anti-PDGFR-beta neutralizing antibody significantly blocks PDGF-AB/FGF-2-induced vessel stability. These findings demonstrate that PDGFR-beta receptor is essential for vascular stability. Similarly, PDGF-AB/FGF-2 significantly induces stable collateral growth in the rat ischemic hind limb. The high number of collaterals induced by PDGF-AB/FGF-2 leads to dramatic improvement of the paw's skin perfusion. Immunohistochemical analysis of the treated skeletal muscles confirms that a combination of PDGF-AB and FGF-2 significantly induces arteriogenesis in the ischemic tissue. A combination of PDGF-AB and FGF-2 would be optimal proangiogenic agents for the treatment of ischemic diseases.

Purpose: Low molecular weight carbonyl compounds, such as the alpha-ketoaldehydes methylglyoxal (MGO) and glyoxal (GO), are formed under hyperglycemic conditions and behave as advanced glycation end product (AGE) precursors. They form adducts on proteins, thereby inducing cellular dysfunctions involved in chronic complications of diabetes., Methods and Main Findings: Nontoxic concentrations of GO or MGO altered the PDGF-induced PDGFRbeta-phosphorylation, ERK1/2-activation, and nuclear translocation, and the subsequent proliferation of mesenchymal cells (smooth muscle cells and skin fibroblasts). This resulted mainly from inhibition of the intrinsic tyrosine kinase of PDGFRbeta and in part from altered PDGF-BB binding to PDGFRbeta. Concomitantly, the formation of AGE adducts (N(epsilon)carboxymethyl-lysine and N(epsilon)carboxyethyl-lysine) was observed on immunoprecipitated PDGFRbeta. Arginine and aminoguanidine, used as carbonyl scavengers, reversed the inhibitory effect and the formation of AGE adducts on PDGFRbeta. AGE-PDGFRbeta adducts were also detected by anti-AGE antibodies in PDGFRbeta immunopurified from aortas of diabetic (streptozotocin-treated) compared to nondiabetic apolipoprotein E-null mice. Mass spectrometry analysis of aortas demonstrated increased AGE formation in diabetic specimens., Conclusions: These data indicate that MGO and GO induce desensitization of PDGFRbeta that helps to reduce mesenchymal cell proliferation.

Serotonin (5-HT) stimulates smooth muscle cell growth through 5-HT receptors and the 5-HT transporter (5-HTT), and has been associated with pulmonary hypertension (PH). Platelet-derived growth factor receptors (PDGFR) have also been associated with PH. We present evidence for the first time that 5-HT transactivates PDGFRbeta through the 5-HTT in pulmonary artery (PA) SMCs. Inhibition of PDGFR kinase with imatinib or AG1296 blocks 5-HT-stimulated PDGFRbeta phosphorylation. 5-HTT inhibitors and the Na+/K+-ATPase inhibitor ouabain, but not 5-HT2 and 5-HT1B/1D receptor inhibitors, block PDGFRbeta activation by 5-HT. Notably, 5-HTT binds the PDGFRbeta upon 5-HT stimulation and the 5-HTT inhibitor fluoxetine blocks both the binding and PDGDRbeta activation. Activation of PDGFRbeta may occur through oxidation of a catalytic cysteine of tyrosine phosphatase. 5-HT-activated PDGFRbeta phosphorylation is blocked by the antioxidant N-acetyl-L-cysteine and the NADPH oxidase inhibitor, DPI. Inhibition of PDGFR kinase with imatinib or AG1296 significantly inhibits SMC proliferation and migration induced by 5-HT in vitro. Infusion of 5-HT by miniosmotic pumps enhances PDGFRbeta activation in mouse lung in vivo. In summary, these results demonstrate that 5-HT transactivates PDGFRbeta in PASMCs leading to SMC proliferation and migration, and may be an important signaling pathway in the production of PH in vivo.

Destruction of existing tumor blood vessels may be achieved by targeting vascular endothelial growth factor (VEGF) signaling, which mediates not only endothelial cell proliferation but also endothelial cell survival. In this study, however, intravital microscopy failed to demonstrate that targeting of VEGFR-2 (by the tyrosine kinase inhibitor SU5416) induces significant regression of experimental tumor blood vessels. Immunohistochemistry, electron microscopy, expression analyses, and in situ hybridization provide evidence that this resistance of tumor blood vessels to VEGFR-2 targeting is conferred by pericytes that stabilize blood vessels and provide endothelial cell survival signals via the Ang-1/Tie2 pathway. In contrast, targeting VEGFR-2 plus the platelet-derived growth factor receptor (PDGFR)-beta system (PDGFR-beta) signaling (by SU6668) rapidly forced 40% of tumor blood vessels into regression, rendering these tumors hypoxic as shown by phosphorescence quenching. TUNEL staining, electron microscopy, and apoptosis blocking experiments suggest that VEGFR-2 plus PDGFR-beta targeting enforced tumor blood vessel regression by inducing endothelial cell apoptosis. We further show that this is achieved by an interference with pericyte-endothelial cell interaction. This study provides novel insights into the mechanisms of how 1) pericytes may provide escape strategies to anti-angiogenic therapies and 2) novel concepts that target not only endothelial cells but also pericyte-associated pathways involved in vascular stabilization and maturation exert potent anti-vascular effects.

In neurons, hypoxia activates intracellular death-related pathways, yet the antiapoptotic mechanisms triggered by hypoxia remain unclear. In RN46A neuronal cells, minimum media growth conditions induced cell death as early as 12 h after the cells were placed in these conditions (i.e., after removal of B-27 supplement). However, apoptosis occurred in hypoxia (1% O2) only after 48 h, and in fact hypoxia reduced the apoptosis associated with trophic factor withdrawal. Furthermore, hypoxia induced time-dependent increases in expression of platelet-derived growth factor (PDGF) B mRNA and protein, as well as PDGF-beta receptor phosphorylation. Although exogenous PDGF-BB induced only transient Akt activation, hypoxia triggered persistent activation of Akt for up to 24 h. Inhibition of phosphatidylinositol 3-kinase (PI3K) or of PDGF-beta receptor phosphorylation abrogated both hypoxia-induced and exogenous PDGF-BB-induced Akt phosphorylation, and it completely abolished hypoxia-induced protection from media supplement deprivation, which suggests that the long-lasting activation of Akt during hypoxia and the prosurvival induction were due to endogenously generated PDGF-BB. Furthermore, these inhibitors decreased hypoxia-inducible factor 1alpha (HIF-1alpha) DNA binding, which suggests that the PDGF/PDGF-beta receptor/Akt pathway induces downstream HIF-1alpha gene transcription. We conclude that in RN46A neuronal cells, hypoxia activates an autocrine-paracrine antiapoptotic mechanism that involves up-regulation of PDGF-B and PDGF-beta receptor-dependent activation of the PI3K/Akt signaling pathway to induce downstream transcription of survival genes.

RGS proteins finely tune heterotrimeric G-protein signaling. Implying the need for such fine-tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E12.5-E17.5 mouse embryos. The distribution and location of the Rgs5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-beta. Both E14.5 PDGFR-beta- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Gi(alpha) and Gq(alpha) and it attenuated angiotensin II-, endothelin-1-, sphingosine-1-phosphate-, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-beta and GPCR-mediated signaling pathways active during fetal vascular maturation.

Abnormal proliferation of vascular smooth muscle cells (VSMC) as well as the platelet-derived growth factor (PDGF) plays an important role in the development of proliferative cardiovascular diseases. In this study, we show that treatment of rat and human aortic VSMC with 50 microM 2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol (catechin) and epicatechin (EC) fails to inhibit the PDGF-Rb-activated intracellular signal transduction pathway and VSMC growth. In contrast, 10-50 microM epigallocatechin-3 gallate (EGCG), epicatechin-3 gallate (ECG), and catechin-3 gallate (CG), which all have a galloyl group in the 3-position of the catechin structure, effectively inhibit tyrosine-phosphorylation of PDGF-Rb, PI 3'-K, and PLC-gamma1 as well as the PDGF-BB-induced increase in [Ca2+]i. The PDGF-BB-induced increase in DNA synthesis and cell number was inhibited by ECG, EGCG, and CG, but not by catechin and EC. Epigallocatechin (EGC) that has a galloyl group in the 2-position effectively inhibited VSMC growth without affecting the PDGF-Rb signal pathway. A reduction of 45% and 70% of the intimal and medial cell number in the S-phase, respectively, has been observed in the catheter-injured left carotid artery 7 days after treatment of Wistar Kyoto rats with 10 mg/day EGCG. These results suggest that the galloyl group in the P3-position of the catechin structure is essential for inhibiting the PDGF-Rbeta-mediated intracellular signal transduction pathway.