Growth factors (GF) are important in several stages of the pathogenesis of age-related macular disease (AMD). In choroidal neovascularization (CNV) in exudative AMD, the GF involved are similar to those involved in wound healing of the skin. Like granulation tissue of skin, CNV is characterized by clotting, inflammation, angiogenesis and fibrosis, and like in skin wounds, members of the VEGF, angiopoietin, PDGF and TGF-beta families of GF are expressed. However, several of these GF may also serve physiological functions in the normal eye, where the retinal pigment epithelium (RPE) employs them to provide trophic support to the neuroretina and choriocapillaris, in addition to maintaining an anti-angiogenic state. Derangement of these physiological functions may underlie the initiation of CNV in AMD. Basolateral secretion of VEGF-A by the RPE maintains the choriocapillaris, and is enhanced by hypoxia. Age-related changes in Bruch's membrane lead to impairment of this trophic function and choriocapillaris atrophy, as well as to decreased diffusion of oxygen towards the neuroretina. The resulting outer retina hypoxia may be an important driving force of CNV formation, by stimulating VEGF overexpression by the RPE, in addition to the effects of increased oxidative stress and low-grade inflammation. RPE senescence and hypoxia may also decrease expression of angiogenesis inhibitors such as PEDF, further shifting the balance to a pro-angiogenic state in the aging eye.

BACKGROUND

Pericytes are integral members of the neurovascular unit. Using mouse models lacking endothelial-secreted platelet derived growth factor-B (PDGF-B) or platelet derived growth factor receptor beta (PDGFRβ) on pericytes, it has been demonstrated that PDGF-B/PDGFRβ interactions mediate pericyte recruitment to the vessel wall in the embryonic brain regulating the development of the cerebral microcirculation and the blood-brain barrier (BBB). Relatively little is known, however, about the roles of PDGF-B/PDGFRβ interactions and pericytes in the adult brain in part due to a lack of adequate and/or properly characterized experimental models. To address whether genetic disruption of PDGFRβ signaling would result in a pericyte-specific insult in adult mice, we studied the pattern and cellular distribution of PDGFRβ expression in the brain in adult control mice and F7 mice that express two hypomorphic Pdgfrβ alleles containing seven point mutations in the cytoplasmic domain of PDGFRβ that impair downstream PDGFRβ receptor signaling.

RESULTS

Using dual fluorescent in situ hybridization, immunofluorescent staining for different cell types in the neurovascular unit, and a fluorescent in situ proximity ligation assay to visualize molecular PDGF-B/PDGFRβ interactions on brain tissue sections, we show for the first time that PDGFRβ is exclusively expressed in pericytes, and not in neurons, astrocytes or endothelial cells, in the adult brain of control 129S1/SvlmJ mice. PDGFRβ co-localized only with well-established pericyte markers such as Chondroitin Sulfate Proteoglycan NG2 and the xLacZ4 transgenic reporter. We next confirm pericyte-specific PDGFRβ expression in the brains of F7 mutants and show that these mice are viable in spite of substantial 40-60% reductions in regional pericyte coverage of brain capillaries.

CONCLUSIONS

Our data show that PDGFRβ is exclusively expressed in pericytes in the adult 129S1/Sv1mJ and F7 mouse brain. Moreover, our findings suggest that genetic disruption of PDGFRβ signaling results in a pericyte-specific insult in adult F7 mutants and will not exert a primary effect on neurons because PDGFRβ is not expressed in neurons of the adult 129S1/SvlmJ and F7 mouse brain. Therefore, mouse models with normal and deficient PDGFRβ signaling on a 129S1/SvlmJ background may effectively be used to deduce the specific roles of pericytes in maintaining the cerebral microcirculation and BBB integrity in the adult and aging brain as well as during neurodegenerative and brain vascular disorders.

Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The pY542TNI sequence conforms to the consensus binding site for the SH2 domain of Grb2, which, by association with Sos1, couples some growth factor receptors to Ras. Following PDGF stimulation, Grb2 binds tyrosine-phosphorylated SHPTP2. Moreover, a mutant PDGFR lacking its SHPTP2 binding site displays markedly reduced Grb2 binding. These data indicate that phosphorylation of SHPTP2 couples Grb2 to PDGFR in vivo, providing a mechanism for Ras activation by PDGFR and for positive signaling via SHPTP2 and Csw.

It has recently been shown that some growth factors (GFs) have strong interactions with nonproteoglycan extracellular matrix proteins. Relevant here, the 12th-14th type three repeats of fibronectin (FN III12-14) have been shown to bind insulin-like growth factor binding-protein-3, fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF)-A with high affinity. Since FN III12-14 is known to bind GFs from different families, we hypothesized that this domain could be highly promiscuous in its GF-binding capacity. We used biochemical approaches and surface plasmon resonance to investigate such interactions with recombinant FN III12-14. We found that FN III12-14 binds most of the GFs from the platelet-derived growth factor (PDGF)/VEGF and FGF families and some GFs from the transforming growth factor-β and neurotrophin families, with K(D) values in the nanomolar range, without inhibiting GF activity. Overall, 25 new binding interactions were identified. In a clinically relevant fibrin matrix, a fibrin-binding variant of FN III12-14 was highly effective as a GF delivery system. For instance, in matrices functionalized with FN III12-14, PDGF-BB-induced sprouting of human smooth muscle cell spheroids was greatly enhanced. We show that FN III12-14 is a highly promiscuous ligand for GFs and also holds great potential in clinical healing applications.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder characterized by vascular malformations. Many affected individuals develop recurrent nosebleeds, which can severely affect their quality of life and are clinically difficult to treat. We report here that treatment with thalidomide reduced the severity and frequency of nosebleeds (epistaxis) in the majority of a small group of subjects with HHT tested. The blood hemoglobin levels of the treated individuals rose as a result of reduced hemorrhage and enhanced blood vessel stabilization. In mice heterozygous for a null mutation in the Eng gene (encoding endoglin), an experimental model of HHT, thalidomide treatment stimulated mural cell coverage and thus rescued vessel wall defects. Thalidomide treatment increased platelet-derived growth factor-B (PDGF-B) expression in endothelial cells and stimulated mural cell activation. The effects of thalidomide treatment were partially reversed by pharmacological or genetic interference with PDGF signaling from endothelial cells to pericytes. Biopsies of nasal epithelium from individuals with HHT treated or not with thalidomide showed that similar mechanisms may explain the effects of thalidomide treatment in humans. Our findings demonstrate the ability of thalidomide to induce vessel maturation, which may be useful as a therapeutic strategy for the treatment of vascular malformations.

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.

The vascular endothelial growth factor (VEGF) family has recently been expanded by the isolation of two additional growth factors, VEGF-B and VEGF-C. Here we compare the regulation of steady-state levels of VEGF, VEGF-B and VEGF-C mRNAs in cultured cells by a variety of stimuli implicated in angiogenesis and endothelial cell physiology. Hypoxia, Ras oncoprotein and mutant p53 tumor suppressor, which are potent inducers of VEGF mRNA did not increase VEGF-B or VEGF-C mRNA levels. Serum and its component growth factors, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) as well as transforming growth factor-beta (TGF-beta) and the tumor promoter phorbol myristate 12,13-acetate (PMA) stimulated VEGF-C, but not VEGF-B mRNA expression. Interestingly, these growth factors and hypoxia simultaneously downregulated the mRNA of another endothelial cell specific ligand, angiopoietin-1. Serum induction of VEGF-C mRNA occurred independently of protein synthesis; with an increase of the mRNA half-life from 3.5 h to 5.5-6 h, whereas VEGF-B mRNA was very stable (T 1/2>8 h). Our results reveal that the three VEGF genes are regulated in a strikingly different manner, suggesting that they serve distinct, although perhaps overlapping functions in vivo.

The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22(phox)-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-beta(1) (TGF-beta(1)) upregulated HIF-1alpha protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1alpha as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1alpha, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22(phox) antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1alpha, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22(phox)-containing NADPH oxidase is crucially involved in this response.

'Vascular endothelial growth factor-A (VEGF-A) blockade has been recently validated as an effective strategy for the inhibition of new blood vessel growth in cancer and ocular pathologies. However, several studies have also shown that anti-VEGF therapy may not be as effective in the treatment of established unwanted blood vessels, suggesting they may become less dependent on VEGF-A for survival. The VEGF-A dependence of vessels may be related to the presence of vascular mural cells (pericytes or smooth muscle cells). Mural cell recruitment to the growing endothelial tube is regulated by platelet-derived growth factor-B (PDGF-B) signaling, and interference with this pathway causes disruption of endothelial cell-mural cell interactions and loss of mural cells. We have investigated the basis of blood vessel dependence on VEGF-A in models of corneal and choroidal neovascularization using a combination of reagents (an anti-VEGF aptamer and an anti-PDGFR-beta antibody) to inhibit both the VEGF-A and PDGF-B signaling pathways. We demonstrate that neovessels become refractory to VEGF-A deprivation over time. We also show that inhibition of both VEGF-A and PDGF-B signaling is more effective than blocking VEGF-A alone at causing vessel regression in multiple models of neovascular growth. These findings provide insight into blood vessel growth factor dependency and validate a combination therapy strategy for enhancing the current treatments for ocular angiogenic disease.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.

Platelet-derived growth factor (PDGF) was found to induce dimerization of purified B-type PDGF receptors, as analyzed by sodium dodecyl sulfate gel electrophoresis after covalent cross-linking using disuccinimidyl suberate. PDGF-BB was 20-fold more effective than PDGF-AB; PDGF-AA was without effect. The dimerization was dose-dependent and was maximal at 0.5-2 micrograms/ml PDGF-BB; at higher concentrations dimerization was less abundant. This indicates that dimerization occurred when one PDGF-BB molecule bound two receptor molecules. The dimerization correlated to activation of the tyrosine kinase of the receptor, determined as autophosphorylation, but was not dependent on phosphorylation reactions because it occurred also in the absence of ATP. Furthermore, dimerization of the receptor correlated with the ability to phosphorylate phosphofructokinase, an exogenous substrate. The complex of ligand and receptor dimer was stable; it resisted electrophoresis under nondenaturing conditions, as well as gel chromatography. The present data indicate that intermolecular mechanisms are involved in signal transduction from the external ligand binding domain to the internal effector domains of the B-type PDGF receptor.

Immunostaining with endothelial and pericyte markers was used to evaluate the cellular composition of angiogenic sprouts in several types of tumors and in the developing retina. Confocal microscopy revealed that, in addition to conventional endothelial tubes heavily invested by pericytes, all tissues contained small populations of endothelium-free pericyte tubes in which nerve/glial antigen 2 (NG2) positive, platelet-derived growth factor beta (PDGF beta ) receptor-positive perivascular cells formed the lumen of the microvessel. Perfusion of tumor-bearing mice with FITC-dextran, followed by immunohistochemical staining of tumor vasculature, demonstrated direct apposition of pericytes to FITC-dextran in the lumen, confirming functional connection of the pericyte tube to the circulation. Transplantation of prostate and mammary tumor fragments into NG2-null mice led to the formation of tumor microvasculature that was invariably NG2-negative, demonstrating that pericytes associated with tumor microvessels are derived from the host rather than from the conversion of tumor cells to a pericyte phenotype. The existence of pericyte tubes reflects the early participation of pericytes in the process of angiogenic sprouting. The ability to study these precocious contributions of pericytes to neovascularization depends heavily on the use of NG2 and PDGF beta -receptor as reliable early markers for activated pericytes.

The primary structure of the human A-type receptor for platelet-derived growth factor (PDGF) has been determined. A 6.5-kilobase (kb) transcript was identified through low-stringency hybridization with a probe derived from the B-type PDGF receptor cDNA. The sequence of a cDNA clone corresponding to the 6.5-kb transcript contains an open reading frame that predicts a 1089-amino acid growth factor receptor-like molecule, which displays 44% overall amino acid similarity with the PDGF B-type receptor. The two receptors have a similar domain organization, with five immunoglobulin-like domains extracellularly and an intracellular split protein tyrosine kinase domain. Transfection of the new cDNA into COS cells led to the expression of a protein specifically recognized by an antiserum previously shown to react with the PDGF A-type receptor. The expressed protein was shown to display high-affinity binding of all three 125I-labeled dimeric forms of PDGF A and B chains in a manner that is characteristic for the PDGF A-type receptor.

There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbetaPDGF treatment also markedly reduced the TGFbetaPDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbetabetaPDGF treatment of keratocytes reverse the RGD blockade of TGFbetabeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbetaPDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.

Gliomas are primary brain tumors mainly affecting adults. The cellular origin is unknown. The recent identification of tumor-initiating cells in glioma, which share many similarities with normal neural stem cells, has suggested the cell of origin to be a transformed neural stem cell. In previous studies, using the RCAS/tv-a mouse model, platelet-derived growth factor B (PDGF-B)-induced gliomas have been generated from nestin or glial fibrillary acidic protein-expressing cells, markers of neural stem cells. To investigate if committed glial progenitor cells could be the cell of origin for glioma, we generated the Ctv-a mouse where tumor induction would be restricted to myelinating oligodendrocyte progenitor cells (OPCs) expressing 2',3'-cyclic nucleotide 3'-phosphodiesterase. We showed that PDGF-B transfer to OPCs could induce gliomas with an incidence of 33%. The majority of tumors resembled human WHO grade II oligodendroglioma based on close similarities in histopathology and expression of cellular markers. Thus, with the Ctv-a mouse we have showed that the cell of origin for glioma may be a committed glial progenitor cell.

Glomerulosclerosis, a final common lesion of various glomerular diseases, is characterized by mesangial cell proliferation and extracellular matrix (ECM) expansion. TGF-beta and PDGF are known to play a critical role in the regulation of ECM metabolism and mesenchymal cell proliferation, respectively. However, there is little evidence to demonstrate the direct role of each of these growth factors in the pathogenesis of glomerulosclerosis. Using an in vivo transfection technique, we could realize the selective overexpression of single growth factor in the kidney. The introduction of either TGF-beta or PDGF-B gene alone into the kidney induced glomerulosclerosis, although the patterns of action of these growth factors were different; TGF-beta affected ECM accumulation rather than cell proliferation and PDGF affected the latter rather than the former.

Platelet concentrates for surgical topical applications are nowadays often used, but quantification of the long-term growth factor release from these preparations in most cases is impossible. Indeed, in most protocols, platelets are massively activated and there is no significant fibrin matrix to support growth factor release and cell migration. Choukroun's platelet-rich fibrin (PRF), a second generation platelet concentrate, is a leucocyte- and platelet-rich fibrin biomaterial. Here, we show that this dense fibrin membrane releases high quantities of three main growth factors (Transforming Growth Factor b-1 (TGFbeta-1), platelet derived growth factor AB, PDGF-AB; vascular endothelial growth factor, VEGF) and an important coagulation matricellular glycoprotein (thrombospondin-1, TSP-1) during 7 days. Moreover, the comparison between the final released amounts and the initial content of the membrane (after forcible extraction) allows us to consider that the leucocytes trapped in the fibrin matrix continue to produce high quantities of TGFbeta-1 and VEGF during the whole experimental time.

Blood platelets become activated and aggregate at the site of vessel injury. Upon activation by thrombin, platelets release storage pools of proteins and growth factors (GFs), including those involved in tissue repair. Our goal was to evaluate the potential beneficial effect of proteins released from platelet-rich clots on tendon healing. PDGF, TGF-beta-1, IGF-I, HGF, VEGF and EGF were measured in human platelet-poor plasma (PPP) and in the releasates collected from either platelet-poor or platelet-rich clots prepared in vitro. We then studied the effects of the releasates on human tendon cells in culture. Releasates from both platelet-rich and platelet-poor clots stimulated tendon cell proliferation, in contrast to un-clotted PPP. The mitogenic activity of the supernatants was not decreased by the thrombin inhibitor, hirudin. Cultured tendon cells synthesise VEGF and HGF in the presence of PPP-clots and PRP-clot releasates, thus the synthesised amount was significantly higher with supernatants from platelet-rich clots than supernatants from a platelet-poor clot (p < 0.05). These results suggest that administering autologous platelet-rich clots may be beneficial to the treatment of tendon injuries by inducing cell proliferation and promoting the synthesis of angiogenic factors during the healing process.

BACKGROUND

Hypoxia and indirect angiogenic factors may stimulate angiogenesis via induction of endothelial cell mitogen(s). To evaluate this hypothesis, we investigated whether low oxygen tension or cytokines known to promote neovascularization in vivo could modulate the expression of either vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) in human vascular smooth muscle cells (SMCs).

RESULTS

SMCs were treated with platelet-derived growth factor BB (PDGF-BB) or transforming growth factor-beta 1 (TGF-beta 1) or exposed to low oxygen tension in serum-free medium. Northern analysis detected low basal levels of VEGF and bFGF mRNA in extracts of unstimulated SMCs. However, both VEGF and bFGF transcripts increased after administration of PDGF-BB (10 or 20 ng/mL) or TGF-beta 1 (0.1 to 10 ng/mL). Hypoxia was a potent stimulus for VEGF gene expression but had no apparent effect on bFGF steady-state mRNA levels.

CONCLUSIONS

These results indicate that certain indirect angiogenic cytokines, such as PDGF-BB or TGF-beta 1, may act via induction of bFGF and VEGF gene expression in cells resident near endothelial cells in vivo. Hypoxia constitutes a potent stimulus for VEGF gene expresion but does not regulate bFGF under the same experimental conditions.

Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation, and increased metabolism of primarily connective tissue cells. In a survey of normal tissues, we found specific immunostaining for PDGF B-chain in neurons, principal dendrites, some axons, and probable terminals throughout the brain, in the dorsal horn of the spinal cord, and in the posterior pituitary of a nonhuman primate (Macaca nemestrina). PDGF activity was extracted from brain cortex and posterior pituitary, and ubiquitous expression of transcripts for the two chains of PDGF and both PDGF receptors was detected throughout the brain and posterior pituitary. A transgenic model was also evaluated in which the chloramphenicol acetyltransferase gene was placed under transcriptional control of the PDGF B-chain promoter. The transgene was preferentially expressed within neural cell bodies in the cortex, hippocampus, and cerebellum. PDGF may act as a neuronal regulatory agent. Neuronal release of PDGF could contribute to nerve regeneration and to glial proliferation that leads to gliosis and scarring.

Smooth muscle cells (SMC) in rat carotid artery leave the quiescent state and proliferate after balloon catheter injury, but the signals for mitogenesis are not known. In this study, the possibility that cells within damaged arteries produce a growth factor that could act locally to stimulate SMC replication and repair was examined. We found that the genes for PDGF-A and -B (ligand) and PDGF receptor (alpha and beta subunits) were expressed in normal and injured carotid arteries and were independently regulated during repair of carotid injury. Two phases of PDGF ligand and receptor gene expression were observed: (a) In the early stage, a large decrease in PDGF beta-receptor mRNA levels preceded 10- to 12-fold increases in PDGF-A transcript abundance in the first 6 h after wounding. No change in PDGF alpha-receptor or PDGF-B gene expression was found at these times. (b) In the chronic phase, 2 wk after injury, neointimal tissue had lower levels of PDGF alpha-receptor mRNA (threefold) and higher levels of PDGF beta-receptor mRNA (three- to fivefold) than did restored media. Moreover, in situ hybridization studies identified a subpopulation of neointimal SMC localized at or near the luminal surface with a different pattern of gene expression than the underlying carotid SMC. Luminal SMC were strongly positive for PDGF-A and PDGF beta-receptor transcripts, while showing little or no hybridization for PDGF-B or PDGF alpha-receptor. Immunohistochemical studies showed strongly positive staining for PDGF-A in SMC along the luminal surface. These data show that changes in PDGF ligand and receptor expression occur at specific times and locations in injured carotid artery and suggest that these changes may play a role in regulating arterial wound repair.

BACKGROUND

In the present series of experiments, we examined the onset of cell proliferation and growth factor expression after balloon overstretch injury to porcine coronary arteries.

RESULTS

Domestic juvenile swine underwent balloon overstretch injury to the left anterior descending and circumflex coronary arteries with standard percutaneous transluminal coronary angioplasty balloon catheters. To identify proliferating cells, 5-bromo-2-deoxyuridine (BrDU) was administered over a period of 24 hours before the animals were killed at either 1, 3, 7, or 14 days after injury. Immunohistochemistry was performed with monoclonal antibodies to BrDU and smooth muscle cell markers. Three days after injury, a large number of proliferating cells were located in the adventitia, with significantly fewer positive cells found in the media and lumen. Seven days after injury, proliferating cells were found primarily in the neointima, extending along the luminal surface. In situ hybridization for PDGF A-chain and beta-receptor mRNAs revealed that the expression of these two genes was closely correlated with the sites of proliferation at each time point. Studies in which BrDU was injected between days 2 and 3 and the animals were killed on day 14 suggested that the proliferating adventitial cells may migrate into the neointima.

CONCLUSIONS

These data suggest that adventitial myofibroblasts contribute to the process of vascular lesion formation by proliferating, synthesizing growth factors, and possibly migrating into the neointima. Increased synthesis of alpha-smooth muscle actin observed in the adventitial cells after arterial injury may constrict the injured vessel and contribute to the process of arterial remodeling and late lumen loss after angioplasty.

This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF beta-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF alpha- and beta-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF beta-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF beta-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF beta-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF beta-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF alpha- and beta-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF beta-receptor expression significantly correlates with less favorable clinicopathological parameters and shorter survival. These findings highlight the prognostic significance of stromal markers and should be considered in ongoing clinical development of PDGF receptor inhibitors.

Vascular endothelial cells, by virtue of their unique anatomical position, are constantly exposed to the fluid mechanical forces generated by flowing blood. In vitro studies with model flow systems have demonstrated that wall shear stresses can modulate various aspects of endothelial structure and function. Certain of these effects appear to result from the regulation of expression of endothelial genes at the transcriptional level. Recent molecular biological studies have defined a "shear stress response element" (SSRE) in the promoter of the human platelet-derived growth factor (PDGF)-B chain gene that interacts with DNA binding proteins in the nuclei of shear-stressed endothelial cells to up-regulate transcriptional activity. Insertion of this element into reporter genes also renders them shear-inducible. Further characterization of this and other positive (and negative) shear-responsive genetic regulatory elements, as well as their transactivating factors, should enhance our understanding of vascular endothelium as an integrator of humoral and biomechanical stimuli in health and disease.