Impaired autoregulation in chronic kidney disease can result in elevation of glomerular capillary pressure and progressive glomerular damage; however, the factors linking chronic glomerular disorders to impaired autoregulation have not been identified. We tested the hypothesis that the cytokine most closely associated with progressive glomerular disease, transforming growth factor (TGF)-beta, may also attenuate autoregulation. Kidneys from normal rats were prepared for videomicroscopy, using the blood-perfused juxtamedullary nephron technique. Autoregulatory responses were measured under control conditions and during superfusion with TGF-beta1 (10 ng/ml). Control afferent arteriolar diameter averaged 18.4 +/- 1 microm and significantly decreased to 16.3 +/- 0.9 and 13.2 +/- 0.8 microm at perfusion pressures of 130 and 160 mmHg, respectively. In the presence of TGF-beta1, autoregulatory responses were completely blocked. In similar experiments performed using PDGF-BB (10 ng/ml) and HGF (25 ng/ml), the normal autoregulatory response was not affected. In vitro studies, using isolated preglomerular vascular smooth muscle cells, revealed that exposure to TGF-beta1 stimulated a rapid increase in reactive oxygen species (ROS) that was inhibited by NADPH oxidase inhibitors. In situ studies, with dihydroethidium staining, revealed a marked increase in renal vessel ROS production on exposure to TGF-beta1. Pretreatment of the juxtamedullary afferent arterioles with tempol, a ROS scavenger, or with apocynin, a NADPH oxidase inhibitor, prevented the impaired autoregulation induced by TGF-beta1. These data reveal a novel hemodynamic pathway by which TGF-beta could lead to progressive glomerular injury by impairing normal renal microvascular function.

Microvascular endothelial cells in vivo exhibit a plastic phenotype, forming a nonproliferative, differentiated capillary network, while retaining their ability to respond to injury by proliferation, migration and neovascularization. The presence of PDGF receptors and PDGF responsiveness in microvascular endothelial cells and the significance of PDGF isoforms in the control of endothelial cell growth and differentiation remain controversial. Since culture of microvascular endothelial cells in a three-dimensional (3D) system induced cell differentiation and angiogenesis and inhibited proliferation, the present study investigates the role of different extracellular matrix environments in inducing different microvascular endothelial cell phenotypes on microvascular endothelial cell PDGF receptor expression and PDGF responsiveness. In conventional two-dimensional (2D) culture, microvascular endothelial cells expressed both PDGF receptor alpha and beta chains. Suramin treatment demonstrated continuous downregulation of the alpha receptor surface expression. PDGF BB and, to a lesser extent, PDGF AB were mitogenic in 2D-culture, PDGF AA failed to induce any proliferative response despite inducing receptor autophosphorylation. During in vitro angiogenesis induced by 3D-culture, both PDGF receptors were rapidly downregulated. Assessment of cell proliferation showed quiescent cells and PDGF unresponsiveness. We conclude that the induction of a differentiated phenotype during in vitro angiogenesis (tube formation) driven in part by the spatial organization of the surrounding matrix is associated with a downregulation of PDGF receptors. Identification of the molecular cell-matrix interactions involved in this receptor regulation may allow for targeted manipulation of cell growth in vivo and lead to novel therapeutic applications for PDGF.

The initiation of de novo testis cord organization in the fetal gonad is poorly understood. Endothelial cell migration into XY gonads initiates testis morphogenesis. However, neither the signals that regulate vascularization of the gonad nor the mechanisms through which vessels affect tissue morphogenesis are known. Here, we show that Vegf signaling is required for gonad vascularization and cord morphogenesis. We establish that interstitial cells express Vegfa and respond, by proliferation, to endothelial migration. In the absence of vasculature, four-dimensional imaging of whole organs revealed that interstitial proliferation is reduced and prevents formation of wedge-like structures that partition the gonad into cord-forming domains. Antagonizing vessel maturation also reduced proliferation. However, proliferation of mesenchymal cells was rescued by the addition of PDGF-BB. These results suggest a pathway that integrates initiation of vascular development and testis cord morphogenesis, and lead to a model in which undifferentiated mesenchyme recruits blood vessels, proliferates in response, and performs a primary function in the morphogenesis and patterning of the developing organ.

Figure 2 summarizes our current interpretation of data concerning signals from the activated PDGF receptor involved in directed migration and proliferation of human arterial SMC. Binding of PDGF (PDGF-BB or PDGF-AA) causes PDGF-receptor dimerization, tyrosine autophosphorylation, and subsequent binding of several molecules containing SH2 domains to the activated receptor. Binding and activation of PLC gamma by the PDGF receptor leads to PIP2 hydrolysis, resulting in generation of diacylglycerol (DAG) and IP3. Subsequently, intracellular levels of calcium are elevated as a result of IP3-mediated calcium release from intracellular compartments. The decreased levels of PIP2 and increased levels of calcium both favor actin-filament disassembly by inducing capping of actin-filament barbed ends and actin-monomer sequestration. A localized, and transient, actin-filament disassembly enables the cell to extend filopodia towards PDGF, thereby enabling chemotaxis to take place. At a later time and/or in a different compartment, actin-filament assembly is promoted by PDGF by a mechanism that is not completely understood, but that may involve small GTP-binding proteins, such as Rho, and formation of DAG. Migration on collagen requires functional alpha 2 beta 1 integrins, which may either constitute a permissive state required for a cell to migrate, or which may be actively involved in intracellular signals leading to migration. PDGF-induced DNA synthesis and proliferation involves activation of Ras, MAP kinase kinase, and MAP kinase. Cross-talk between PKA signaling and tyrosine-kinase receptor signaling results in PKA inhibition of the MAP kinase cascade, probably at the level of Raf. Activation of PI 3-kinase, or a PI 3-kinase-like enzyme, is also likely to contribute to the mitogenic effects of PDGF in these cells (Bornfeldt, unpublished observation). What determines if a SMC will migrate and/or proliferate in response to PDGF? Results are starting to emerge that show regulation of expression of molecules involved in intracellular signaling with different phenotypic states of SMC. For example, expression of PLC gamma is very low in intact vascular wall (where SMC show a "contractile phenotype"), and induced when SMC are converted to a "synthetic phenotype" in culture. Proliferation and expression of MAP kinase, but not calcium signaling, appear to be regulated by the extracellular matrix, and the profile of integrin expression is different in SMC in culture compared to SMC in the vascular wall. Thus, the relation between expression of signaling molecules involved in migration and signaling molecules involved in proliferation, as well as cross-talk between different signal-transduction pathways, may determine the net effect of PDGF.

Growth substances (cytokines and growth factors) are soluble signaling proteins affecting the process of normal wound healing. Cytokines govern the inflammatory phase that clears cellular and extracellular matrix debris. Wound repair is controlled by growth factors (platelet-derived growth factor [PDGF], keratinocyte growth factor, and transforming growth factor beta). Endogenous growth factors communicate across the dermal-epidermal interface. PDGF is important for most phases of wound healing. Becaplermin (PDGF-BB), the only growth factor approved by the Food and Drug Administration, requires daily application for neuropathic wound healing. Gene therapy is under development for more efficient growth factor delivery; a single application will induce constitutive growth factor expression for weeks. Based on dramatic preclinical animal studies, a phase 1 clinical trial planned on a PDGF genetic construct appears promising.

Despite the growing understanding of pdgf signaling, studies of pdgf function have encountered two major obstacles: the functional redundancy of PDGFRalpha and PDGFRbeta in vitro and their distinct roles in vivo. Here we used wild-type mouse embryonic fibroblasts (MEF), MEF null for either PDGFRalpha, beta, or both to dissect PDGF-PDGFR signaling pathways. These four PDGFR genetically defined cells provided us a platform to study the relative contributions of the pathways triggered by the two PDGF receptors. They were treated with PDGF-BB and analyzed for differential gene expression, in vitro proliferation and differential response to pharmacological effects. No genes were differentially expressed in the double null cells, suggesting minimal receptor-independent signaling. Protean differentiation and proliferation pathways are commonly regulated by PDGFRalpha, PDGFRbeta and PDGFRalpha/beta while each receptor is also responsible for regulating unique signaling pathways. Furthermore, some signaling is solely modulated through heterodimeric PDGFRalpha/beta.

BACKGROUND

Local delivery methods can target therapies to specific tissues and potentially avoid toxicity to other organs. Platelet-derived growth factor can protect the myocardium, but it also plays an important role in promoting pulmonary hypertension. It is not known whether local myocardial delivery of platelet-derived growth factor during myocardial infarction (MI) can lead to sustained cardiac benefit without causing pulmonary hypertension.

RESULTS

We performed a randomized and blinded experiment of 127 rats that survived experimental MI or sham surgery. We delivered platelet-derived growth factor (PDGF)-BB with self-assembling peptide nanofibers (NFs) to provide controlled release within the myocardium. There were 6 groups with n>> or = 20 in each group: sham, sham+NF, sham+NF/PDGF, MI, MI+NF, and MI+NF/PDGF. Serial echocardiography from 1 day to 3 months showed significant improvement of ventricular fractional shortening, end-systolic dimension, and end-diastolic dimension with local PDGF delivery (P < 0.05 for MI+NF/PDGF versus MI or MI+NF). Catheterization at 4 months revealed improved ventricular function in the controlled delivery group (left ventricular end-diastolic pressure, cardiac index, +dP/dt, -dP/dt, and time constant of exponential decay all P < 0.05 for MI+NF/P versus MI or MI+NF). Infarcted myocardial volume was reduced by NF/PDGF therapy (34.0 +/- 13.3% in MI, 28.9 +/- 12.9% in MI+NF, and 12.0 +/- 5.8% in MI+NF/PDGF; P < 0.001). There was no evidence of pulmonary toxicity from the therapy, with no differences in right ventricular end-systolic pressure, right ventricular dP/dt, bromodeoxyuridine staining, or pulmonary artery medial wall thickness.

CONCLUSIONS

Intramyocardial delivery of PDGF by self-assembling peptide NFs leads to long-term improvement in cardiac performance after experimental infarction without apparent pulmonary toxicity. Local myocardial protection may allow prevention of heart failure without systemic toxicity.

Cell migration is critical for many processes, such as angiogenesis, inflammation, development and wound healing, and is also involved in tumour progression and metastasis. Here we show that CXCL12, complement factor 5a (C5a), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF)-BB, which stimulate cell migration, also activate p38alpha MAPK. Pharmacological inhibition of this protein kinase with SB 203580 or BIRB 0796, or the genetic ablation of p38alpha MAPK, blocked cell migration induced by the aforementioned chemo-attractants. Macrophages from mice lacking one or more of the other p38 MAPK isoforms showed normal cell migration in response to C5a. We also show that the activation of p38alpha MAPK in response to CXCL12 requires the p21-activated protein kinases (PAK)-1 and PAK-2. MAPKAP-K2 is a protein kinase that is activated by p38alpha MAPK. Reducing its expression using RNA interference blocked CXCL12-induced HeLa cell migration, while macrophages from mice that do not express MAPKAP-K2 failed to migrate in response to C5a. Moreover, RNA interference against the small heat shock protein 27 (HSP27), a physiological substrate of MAPKAP-K2, blocked the CXCL12-induced cell migration. These results demonstrate a general and essential role of the PAK-p38alpha MAPK-MAPKAP-K2-HSP27 signalling pathway in mediating the effects of chemotactic stimuli on cell migration.

Platelet-derived growth factor (PDGF) and insulin-like growth factor I (IGF-I) in combination have previously been shown to enhance periodontal regeneration. The objective of this study was to further characterize the biological effects of this combination of growth factors in non-human primates and compare the effects to those of each growth factor individually. Ligature-induced periodontitis was initiated in 10 cynomolgus monkeys. After periodontal lesions were established, surgery was performed, and either a methylcellulose gel vehicle or vehicle containing 10 micrograms each of either PDGF-BB, IGF-I or both PDGF-BB and IGF-I was applied to exposed root surfaces. Biopsies were taken 4 and 12 wk after treatment and the extent of periodontal regeneration was assessed by histomorphometry. At both 4 and 12 wk vehicle-treated lesions generally revealed minimal osseous defect fill (ODF) (8.5 +/- 2.1% and 14.5 +/- 5.7%, respectively) and new attachment (NA) (34.1 +/- 5.2% and 26.6 +/- 10.5%, respectively). IGF-I treatment did not significantly alter healing compared to vehicle in any parameter at both 4 and 12 wk. PDGF-BB-treated sites exhibited significant (p < 0.05) regeneration of NA (69.6 + 12.0%) at 12 wk; trends for PDGF-BB treatment effect were also observed in other parameters at 4 and 12 wk, although these increases were not statistically significant. Treatment with PDGF-BB/IGF-I resulted in 21.6 +/- 5.1% and 42.5 +/- 8.3% ODF at 4 and 12 wk, respectively, and 64.1 +/- 7.7% and 74.6 +/- 7.4% NA at 4 and 12 wk, respectively (all significantly greater than vehicle, p < 0.05). The results from this study demonstrated that: 1) IGF-I alone at the dose tested did not significantly alter periodontal wound healing; 2) PDGF-BB alone significantly stimulated NA, with trends of effect on other parameters; and 3) the PDGF-BB/IGF-I combination resulted in significant increases in NA and ODF above vehicle at both 4 and 12 wk.

The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells (VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin (FN)-alpha5beta1 interaction on the surface of VSMC using atomic force microscopy (AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN-VSMC adhesions were selectively blocked by antibodies against alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 single-bond strength. The ability of FN to adhere to VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet-derived growth factor (PDGF)-BB, whereas lysophosphatidic acid (LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time-dependent increase in number of adhesions and/or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of alpha5beta1 and focal contact assembly can be rapidly regulated.

Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-β1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM.

In spite of long term clinical use of decellularized bladder submucosa matrix (BSM), little is known about the active factors within this material. In this paper, we analyzed the biological factors from the decellularized BSM using ELISA, Western blotting, and immunohistochemistry for the purpose of effective utilization of this material in the field of regenerative medicine. At least 10 growth factors, including VEGF, BMP4, PDGF-BB, KGF, TGFbeta1, IGF, bFGF, EGF and TGFalpha were found to be preserved in the decellularized BSM. The existence of collagen (type 1, 2, 3, 4), laminin and elastin within the matrix was also demonstrated. The soluble BSM extracts showed a conspicuous effect on cell proliferation when added as a supplement in vitro. These findings demonstrate that growth factors and extracellular matrix in the BSM maintain valuable biological activity even after the decellularization and extraction processes, thus supporting the wide applicability of BSM in tissue regeneration. The identification and characterization of growth factors and extracellular matrix in the BSM is a prerequisite for understanding tissue regeneration using this scaffold.

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.

Growth factor stimulation of quiescent cells induces a series of intracellular early and late events that ultimately lead to DNA synthesis and cell division. We describe here that production of phosphorylcholine is an essential component of the late events involved in the induction of DNA synthesis by platelet-derived growth factor (PDGF-BB), a prototype mitogen for fibroblasts. Moreover, phosphorylcholine itself is mitogenic when added exogenously to NIH3T3 cells, further indicating its role as a crucial intracellular messenger for DNA synthesis. Choline kinase, the first step in the route of phosphatidylcholine synthesis appears to be the critical regulatory enzyme in phosphorylcholine production, indicating that regulation of choline kinase represents a key step during mitogenic stimulation. We also describe that several growth factors (PDGF-AA, basic FGF, EGF and phorbol esters) rely on their ability to generate phosphorylcholine for their proliferating activity. In contrast, DNA synthesis induced by serum did not require phosphorylcholine. Moreover, the requirement for phosphorylcholine production in PDGF-stimulated cells can be over-ruled by addition of insulin. Thus, cell proliferation in NIH3T3 cells can be triggered off by alternative pathways and one of them involves generation of phosphorylcholine.

Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G(1) cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G(1) phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27(Kip1) expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27(Kip1) protein. The time course of p27(Kip1) decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27(Kip1) synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-beta1 by aortic SMC. These results identify p27(Kip1) as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.

The Src family protein-tyrosine kinases are required for mitogenic signaling from the platelet-derived growth factor (PDGF), colony stimulating factor-1, and epidermal growth factor (EGF) receptor protein-tyrosine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A.(1995) Mol. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblasts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activated. Src family catalytic function is required for RPTK mitogenic signaling as evidenced by the SH2-dependent dominant negative phenotype exhibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700). Here, we have generated clonal Src- murine fibroblast cell lines overexpressing various murine c-Src mutants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F, each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent cells. This demonstrates an involvement of the Src SH3 domain in PDGFbeta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mutant on PDGF receptor signaling was reversed by a second SH2-inactivating mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because binding of c-Src to the receptor via its SH2 domain is a prerequisite for the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling.

Several transmembrane molecules are cleaved at juxtamembrane extracellular sites leading to shedding of ectodomains. We analysed shedding of members of the Vps10p-D (Vps10p domain; where Vps is vacuolar protein sorting) family of neuronal type-I receptors with partially overlapping functions, and additional proteolytic events initiated by the shedding. When transfected into CHO (Chinese-hamster ovary) cells (CHO-K1), sorCS1a-sorCS1c isoforms were shed at high rates (approximately 0.61% x min(-1)) that were increased approx. 3-fold upon stimulation with phorbol ester. sorCS1c identified in the cultured neuroblastoma cell line SH-SY5Y was shed similarly. In CHO-K1 transfectants, constitutive and stimulated shedding of sorCS3 also occurred at high rates (0.29% and 1.03% x min(-1)). By comparison, constitutive and stimulated shedding of sorLA occurred at somewhat lower rates (0.07% and 0.48% x min(-1)), whereas sorCS2 and sortilin were shed at very low rates even when stimulated (approximately 0.01% x min(-1)). Except for sorCS2, shedding of the receptors was dramatically reduced in mutant CHO cells (CHO-M2) devoid of active TACE (tumour necrosis factor alpha-converting enzyme), demonstrating that this enzyme accounts for most sheddase activity. The release of sorCS1 and sorLA ectodomains initiated rapid cleavage of the membrane-tethered C-terminal stubs that accumulated only in the presence of gamma-secretase inhibitors. Purified shed sorLA bound several ligands similarly to the entire luminal domain of the receptor, including PDGF-BB (platelet-derived growth factor-BB) and amyloid-beta precursor protein. In addition, PDGF-BB also bound to the luminal domains of sorCS1 and sorCS3. The results suggest that ectodomains shed from a subset of Vps10p-D receptors can function as carrier proteins.

Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We detected little PDGF isoform expression in normal skin and in nonhealing dermal ulcers. In contrast, in surgically created acute wounds and chronic ulcers treated with rPDGF-BB, markedly upregulated levels of PDGF-AA (long form) were found. In both types of wounds, increased PDGF-AA was detected primarily in capillaries and fibroblasts, although in rPDGF-BB-treated chronic wounds, widespread expression of PDGF-AA was somewhat delayed. With continued treatment, the long form of PDGF-AA, which can preferentially bind extracellular matrix, was expressed only in capillaries, while fibroblasts began synthesizing the short form of PDGF-AA. Within capillaries, all endothelial cells and varying numbers of pericytes and smooth muscle cells contained PDGF-AA. In all wounds, macrophages and keratinocytes were not a major contributor. While PDGF-BB and PDGF-AB were present in a minority of healing wounds, they were usually present at lower levels than PDGF-AA. PDGF-beta receptors, which bind only PDGF-BB and not other isoforms, were found in normal skin and granulation tissue, providing a molecular basis for treating human chronic wounds with exogenous rPDGF-BB.

OBJECTIVE

To determine the temporal and spatial relationship between platelet-derived growth factor beta (PDGFbeta) receptors, PDGF-AB/BB, and activated pericytes across the Raynaud's phenomenon (RP) and systemic sclerosis (SSc; scleroderma) disease spectrum.

METHODS

Monoclonal antibodies against PDGFbeta receptors, PDGF-AB/BB, and high molecular weight-melanoma-associated antigen (HMW-MAA), a marker for activated pericytes, were used to immunohistochemically analyze serial sections of skin biopsy tissue from patients with RP and from scleroderma patients. To delineate cell-specific PDGFbeta receptor expression, double immunofluorescence-stained sections were analyzed using computer-aided image analysis and confocal microscopy.

RESULTS

PDGFbeta receptor-expressing cells and HMW-MAA-expressing pericytes were found in biopsy samples from autoimmune RP patients and in both early fibrotic and early nonfibrotic scleroderma skin, but not in normal or primary RP or late-stage scleroderma skin. PDGF-AB/BB was expressed within the epidermis, at the epidermal/dermal junction, and by dermal macrophages. Analysis of juxtaposed serial sections revealed an increased frequency of receptor expression in microvessels from autoimmune RP and early scleroderma skin (P < 0.01). Double-labeling studies using confocal microscopy showed that, in vivo, PDGFbeta receptors were predominantly expressed by microvascular pericytes from both autoimmune RP and early scleroderma skin.

CONCLUSIONS

PDGFbeta receptors are expressed by activated microvascular pericytes in patients with autoimmune RP and in early SSc patients, but not in those with primary RP or late-stage scleroderma. These findings suggest that features of autoimmune RP are distinct from those of primary RP, and that microvascular pericytes may be an important link between chronic microvascular damage and fibrosis.

Using human MSCs (mesenchymal stem cells) lacking VEGF (vascular endothelial growth factor) receptors, we show that the pro-angiogenic receptor neuropilin-1 associates with phosphorylated PDGFRs [PDGF (platelet-derived growth factor) receptors], thereby regulating cell signalling, migration, proliferation and network assembly. Neuropilin-1 co-immunoprecipitated and co-localized with phosphorylated PDGFRs in the presence of growth factors. Neuropilin-1 knockdown blocked PDGF-AA-induced PDGFRalpha phosphorylation and migration, reduced PDGF-BB-induced PDGFRbeta activation and migration, blocked VEGF-A activation of both PDGFRs, and attenuated proliferation. Neuropilin-1 prominently co-localized with both PDGFRs within MSC networks assembled in Matrigel and in the chorioallantoic membrane vasculature microenvironment, and its knockdown grossly disrupted network assembly and decreased PDGFR signalling. Thus neuropilin-1 regulates MSCs by forming ligand-specific receptor complexes that direct PDGFR signalling, especially the PDGFRalpha homodimer. This receptor cross-talk may control the mobilization of MSCs in neovascularization and tissue remodelling.

Perivascular supporting cells, including vascular smooth muscle cells (VSMCs) and pericytes (PCs), provide instructive signals to adjacent endothelial cells helping to maintain vascular homeostasis. These signals are provided through direct contact and by the release of soluble factors by these cells. Thrombospondin (TSP)1 is a matricellular protein and an autocrine factor for VSMCs. TSP1 activity, along with that of PDGF, regulates VSMC proliferation and migration. However, the manner in which TSP1 and PDGF impact retinal PC function requires further investigation. In the present study, we describe, for the first time, the isolation and culture of retinal PCs from wild-type (TSP1(+/+)) and TSP1-deficient (TSP1(-/-)) immortomice. We showed that these cells express early and mature markers of PCs, including NG2, PDGF receptor-beta, and smooth muscle actin as well as desmin, calbindin, and mesenchymal stem cell markers. These cells were successfully passaged and maintained in culture for several months without significant loss of expression of these markers. TSP1(+/+) PCs proliferated at a faster rate compared with TSP1(-/-) PCs. In addition, TSP1(+/+) PCs, like VSMCs, responded to PDGF-BB with enhanced migration and proliferation. In contrast, TSP1(-/-) PCs failed to respond to the promigratory and proliferative activity of PDGF-BB. This may be attributed, at least in part, to the limited interaction of PDGF-BB with TSP1 in null cells, which is essential for PDGF proliferative and migratory action. We observed no significant differences in the rates of apoptosis in these cells. TSP1(-/-) PCs were also less adherent, expressed increased levels of TSP2 and fibronectin, and had decreased amounts of N-cadherin and alpha(v)beta(3)-integrin on their surface. Thus, TSP1 plays a significant role in retinal PC proliferation and migration impacting retinal vascular development and homeostasis.

Pericytes play an important role in modulating angiogenesis, but the origin of these cells is poorly understood. To evaluate whether the mature vessel wall contains pericyte progenitor cells, nonendothelial mesenchymal cells isolated from the rat aorta were cultured in a serum-free medium optimized for stem cells. This method led to the isolation of anchorage-independent cells that proliferated slowly in suspension, forming spheroidal colonies. This process required basic fibroblast growth factor (bFGF) in the culture medium, because bFGF withdrawal caused the cells to attach to the culture dish and irreversibly lose their capacity to grow in suspension. Immunocytochemistry and RT-PCR analysis revealed the expression of the precursor cell markers CD34 and Tie-2 and the absence of endothelial cell markers (CD31 and endothelial nitric oxide synthase, eNOS) and smooth muscle cell markers (alpha-smooth muscle actin, alpha-SMA). In addition, spheroid-forming cells were positive for NG2, nestin, PDGF receptor (PDGFR)-alpha, and PDGFR-beta. Upon exposure to serum, these cells lost CD34 expression, acquired alpha-SMA, and attached to the culture dish. Returning these cells to serum-free medium failed to restore their original spheroid phenotype, suggesting terminal differentiation. When embedded in collagen gels, spheroid-forming cells rapidly migrated in response to PDGF-BB and became dendritic. Spheroid-forming cells cocultured in collagen with angiogenic outgrowths of rat aorta or isolated endothelial cells transformed into pericytes. These results demonstrate that the rat aorta contains primitive mesenchymal cells capable of pericyte differentiation. These immature cells may represent an important source of pericytes during angiogenesis in physiological and pathological processes. They may also provide a convenient supply of mural cells for vascular bioengineering applications.

OBJECTIVE

Growth factor delivery may be useful to accelerate the rate of tendon healing. Before in vivo use, however, the effects of growth factors on tendon cells need to be well characterized. The purpose of this study was to evaluate the effects of 4 growth factors on intrasynovial tendon fibroblast proliferation and collagen production in vitro. Our first hypothesis was that platelet-derived growth factor BB (PDGF-BB) and basic fibroblast growth factor (bFGF) would promote cell proliferation and collagen production. Our second hypothesis was that there would be a positive effect from the combination of PDGF-BB and bFGF.

METHODS

The growth factors PDGF-BB, bFGF, vascular endothelial growth factor (VEGF), and bone morphogenetic protein 2 (BMP-2) were evaluated in vitro with canine flexor tendon fibroblasts. The effects of single factors (PDGF-BB, bFGF, VEGF, or BMP-2) or a combination of factors (PDGF-BB and bFGF) on cell proliferation (ie, thymidine incorporation) and collagen production (ie, proline incorporation) were evaluated.

RESULTS

The results supported our hypotheses. Cell proliferation increased significantly with PDGF-BB and bFGF. Collagen production also increased significantly with PDGF-BB and bFGF. Cell proliferation and collagen production were unchanged with VEGF and BMP-2. A dose-response effect was seen for PDGF-BB combined with bFGF. The combination of PDGF-BB and bFGF led to an increase in cell proliferation but no change in collagen production compared with each factor alone.

CONCLUSIONS

The growth factors PDGF-BB and bFGF significantly increased flexor tendon fibroblast proliferation and matrix synthesis when applied singly. Administration of PDGF-BB and bFGF combined led to increased proliferation to single factors.

The Src homology 2 (SH2) domain-containing Shc proteins p52shc and p46shc become phosphorylated upon activation of several tyrosine kinases and are implicated in mitogenic signal transduction. Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to autophosphorylation of tyrosine residues, which is known to mediate interactions with several SH2 domain-containing signaling molecules. In this study, we have characterized the interaction between the PDGF beta-receptor and Shc. PDGF beta-receptor coprecipitation in Shc immunoprecipitates was dependent on stimulation with PDGF-BB. The Shc SH2 domain expressed as a bacterial fusion protein bound the autophosphorylated PDGF beta-receptor. Moreover, the Shc SH2 domain could bind the autophosphorylated purified baculovirus-expressed PDGF beta-receptor intracellular domain, which indicates a direct association of Shc with the PDGF beta-receptor. Activation of the PDGF beta-receptor induced the preferential phosphorylation of p52shc. Tyrosine-phosphorylated Shc, in turn, formed a complex with the signaling molecule Grb2. Synthetic peptide analysis revealed that certain autophosphorylation sites in the PDGF beta-receptor (Tyr-579, Tyr-740, Tyr-751, and Tyr-771) were able to mediate the specific binding of the Shc SH2 domain as well as intact Shc proteins. A mutant PDGF beta-receptor in which Tyr-579 was replaced with phenylalanine showed 40% impaired association of Shc in vivo, but phosphorylation of Shc proteins was not affected. We conclude that multiple autophosphorylation sites in the PDGF beta-receptor are responsible for the binding of Shc. This is in contrast to previously characterized interactions between the PDGF beta-receptor and SH2 domain-containing proteins, which generally involve one high affinity binding site in the receptor.