The objective of this research is to develop a dual growth factor-releasing nanoparticle-in-nanofiber system for wound healing applications. In order to mimic and promote the natural healing procedure, chitosan and poly(ethylene oxide) were electrospun into nanofibrous meshes as mimics of extracellular matrix. Vascular endothelial growth factor (VEGF) was loaded within nanofibers to promote angiogenesis in the short term. In addition, platelet-derived growth factor-BB (PDGF-BB) encapsulated poly(lactic-co-glycolic acid) nanoparticles were embedded inside nanofibers to generate a sustained release of PDGF-BB for accelerated tissue regeneration and remodeling. In vitro studies revealed that our nanofibrous composites delivered VEGF quickly and PDGF-BB in a relayed manner, supported fibroblast growth and exhibited anti-bacterial activities. A preliminary in vivo study performed on normal full thickness rat skin wound models demonstrated that nanofiber/nanoparticle scaffolds significantly accelerated the wound healing process by promoting angiogenesis, increasing re-epithelialization and controlling granulation tissue formation. For later stages of healing, evidence also showed quicker collagen deposition and earlier remodeling of the injured site to achieve a faster full regeneration of skin compared to the commercial Hydrofera Blue® wound dressing. These results suggest that our nanoparticle-in-nanofiber system could provide a promising treatment for normal and chronic wound healing.

Platelet-derived growth factor (PDGF)-BB, a potent mitogen for mesenchymal cells, also downregulates expression of multiple smooth muscle (SM) cell (SMC)-specific markers. However, there is conflicting evidence whether PDGF-BB represses SMC marker expression at a transcriptional or posttranscriptional level, and little is known regarding the mechanisms responsible for these effects. Results of the present studies provide clear evidence that PDGF-BB treatment strongly repressed SM alpha-actin, SM myosin heavy chain (MHC), and SM22alpha promoters in SMCs. Of major significance for resolving previous controversies in the field, we found PDGF-BB-induced repression of SMC marker gene promoters in subconfluent, but not postconfluent, cultures. Treatment of postconfluent SMCs with a tyrosine phosphatase inhibitor restored PDGF-BB-induced repression, whereas treatment of subconfluent SMCs with a tyrosine kinase blocker abolished PDGF-BB-induced repression, suggesting that a tyrosine phosphorylation event mediates cell density-dependent effects. On the basis of previous observations that Ets-1 transcription factor is upregulated within phenotypically modulated neointimal SMCs, we tested whether Ets-1 would repress SMC marker expression. Consistent with this hypothesis, results of cotransfection experiments indicated that Ets-1 overexpression reduced transcriptional activity of SMC marker promoter constructs in SMCs, whereas it increased activity of SM alpha-actin promoter in endothelial cells. PDGF-BB treatment increased expression of Ets-1 in cultured SMCs, and SM alpha-actin mRNA expression was reduced in multiple independent clones of SMCs stably transfected with an Ets-1-overexpressing construct. Taken together, results of these experiments provide novel insights regarding possible mechanisms whereby PDGF-BB and Ets-1 may contribute to SMC phenotypic switching associated with vascular injury.

Extracellular matrix (ECM) and growth factors are potent regulators of cell phenotype. These biological mediators of cellular responses are potentially interactive and as such could drive cells through progressive phenotypes to create new tissue as in morphogenesis and wound repair. In fact, ECM composition changes during tissue formation accompanied by alterations in cell growth and migration. How alterations in the ECM regulate cell activities is poorly defined. To address this question in wound repair, we cultured normal human dermal skin fibroblasts in relaxed collagen gels, fibronectin-rich cultures or stressed fibrin gels, and stressed collagen gels to model normal dermis, early wound provisional matrix, and late granulation tissue, respectively. Integrin subunits, alpha 2, alpha 3, and alpha 5, that define receptor specificity for collagen and provisional matrix, respectively, were measured at mRNA steady-state level before and after stimulation with platelet-derived growth factor-BB (PDGF-BB), a potent mitogen and chemoattractant for fibroblasts. Fibronectin-rich cultures and fibrin gels supported PDGF-BB induction of alpha 3 and alpha 5 mRNA. In contrast, both stressed and relaxed collagen attenuated these responses while promoting maximal alpha 2 mRNA expression. Posttranscriptional regulation was an important mechanism in this differential response. Together PDGF-BB and collagen gels promoted alpha 2, but not alpha 3 and alpha 5, mRNA stability. Conversely, when fibroblasts were in fibronectin-rich cultures, PDGF-BB promoted alpha 3 and alpha 5, but not alpha 2, mRNA stability. We suggest that ECM alterations during wound healing or any new tissue formation cause cells to respond differently to repeated growth factor stimuli. An ordered progression of cell phenotypes results, ultimately consummating tissue repair or morphogenesis.

Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR) are key mediators of physiological and pathological angiogenesis. They are expressed in most tissues during embryonic development but are down-regulated in the adult, when angiogenesis ceases. Up-regulation of VEGFR-2 and of VEGF are observed in many pathological conditions under which angiogenesis is reinduced. A major regulator of VEGF expression is hypoxia. Although the temporal expression pattern of VEGFR-2 parallels VEGF expression to a high extent, little is known about its regulation. Here, we show that VEGFR-2 is highly expressed in early postnatal mouse brain but is down-regulated commencing at postnatal day 15 (P15) of mouse brain development and is hardly detectable in P30 mouse brain. Using P30 mouse brain slices, we observed that hypoxia up-regulates VEGFR-2 in the slices but not in human umbilical vein endothelial cells, suggesting the presence of a hypoxia-inducible factor in the murine neuroectoderm that up-regulates VEGFR-2. To identify the factors involved, normoxic P30 cerebral slices were cultured with growth factors that are either hypoxia-inducible (e.g., PDGF-BB, erythropoietin, and VEGF) and/or are known to act on endothelial cells (e.g., PDGF-BB, VEGF, and PIGF). Exogenously added recombinant VEGF led to an up-regulation of VEGFR-2 expression, which could be inhibited by preincubation with a neutralizing anti-VEGF antibody. Addition of PDGF-BB, PIGF, and erythropoietin had no effect on VEGFR-2 expression. Our results suggest a differential but synergistic regulation by hypoxia of VEGF and VEGFR-2: a direct induction of VEGF that subsequently up-regulates VEGFR-2 in endothelial cells. This autoenhancing system may represent an important mechanism of tumor angiogenesis.

Porcine aorta endothelial cells are devoid of receptors for platelet-derived growth factor (PDGF). We have transfected such cells with cDNA for the PDGF B-type receptor, both the wild-type receptor and a mutant form of the receptor (K634A), in which the putative nucleotide-binding lysine of the protein-tyrosine domain has been changed to alanine. Immunoprecipitation studies of metabolically labeled cells showed that both types of receptors were synthesized and processed to the mature form of Mr 190,000. In cells expressing the wild-type receptor, PDGF-BB, the natural ligand for the B-type receptor, induced membrane ruffling and reorganization of actin. Such a response has previously been seen in cells expressing the natural PDGF B-type receptor in response to PDGF-BB. No such effect was induced in nontransfected cells or in cells expressing the K634A mutant receptor. PDGF was also shown to be chemotactic for cells expressing the wild-type receptor, whereas no chemotactic response was elicited in control cells or in cells expressing the K634A mutant receptor. Our study thus provides formal evidence that the PDGF B-type receptor mediates a motility response including actin reorganization and chemotaxis. Furthermore, the results establish a role for the receptor-associated protein-tyrosine kinase in the transduction of the chemotactic signal.

The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex.

Smooth muscle cell (SMC) migration and proliferation and extracellular matrix remodeling are essential aspects of the arterial response to injury, vessel development, and atherogenesis. Matrix metalloproteinase (MMP) expression is associated with SMC proliferation and migration after arterial injury. To assess the role of MMPs in SMC proliferation and migration and intimal thickening, we measured the effect of the synthetic MMP inhibitor <em>BB</em>94 (Batimastat) on DNA synthesis and migration of SMCs in vitro as well as the formation of a neointima after balloon injury to the rat carotid artery. <em>BB</em>94 dose-dependently inhibited SMC migration induced by platelet-derived growth factor (<em>PDGF</em>)-<em>BB</em> through a filter coated with a thick basement membrane matrix (Matrigel) layer but did not show any inhibitory effect on SMC migration through a lightly coated filter. At concentrations up to 1 mumol/L, <em>BB</em>94 did not alter DNA synthesis induced by <em>PDGF</em>-AA or <em>PDGF</em>-<em>BB</em>. Treatment with 30 mg <em>BB</em>94.kg-1.d-1 IP for 7 or 14 days after balloon injury to the rat carotid artery decreased the total number of intimal SMC nuclei and suppressed intimal thickening. SMC proliferation (5-bromo-2'-deoxyuridine labeling) was decreased in the media at 2 days, whereas it was increased in the intima at 7 but not 14 days. These results suggest that <em>BB</em>94 inhibits intimal thickening after arterial injury by decreasing SMC migration and proliferation and support the conclusion that MMPs play a significant role in regulating intimal thickening in injured arteries.

Primary and secondary glioblastomas (pGBM, sGBM) are supposed to evolve through different genetic pathways, including EGF receptor and PDGF and its receptor and thus genes that are involved in tumor-induced angiogenesis. However, whether other angiogenic cytokines are also differentially expressed in these glioblastoma subtypes is not known so far, but this knowledge might be important to optimize an antiangiogenic therapy. Therefore, we studied the expression of several angiogenic cytokines, including VEGF-A, HGF, bFGF, PDGF-AB, PDGF-BB, G-CSF and GM-CSF in pGBMs and sGBMs as well as in gliomas WHO III, the precursor lesions of sGBMs. In tumor tissues, expression of all cytokines was observed albeit with marked differences concerning intensity and distribution pattern. Quantification of the cytokines in the supernatant of 30 tissue-corresponding glioma cultures revealed a predominant expression of VEGF-A in pGBMs and significantly higher expression levels of PDGF-AB in sGBMs. HGF and bFGF were determined in nearly all tumor cultures but with no GBM subtype or malignancy-related differences. Interestingly, GM-CSF and especially G-CSF were produced less frequently by tumor cells. However, GM-CSF secretion occurred together with an increased number of simultaneously secreted cytokines and correlated with a worse patient prognosis and may thus represent a more aggressive angiogenic phenotype. Finally, we confirmed an independent contribution of each tumor-derived cytokine analyzed to tumor-induced vascularization. Our data indicate that an optimal antiangiogenic therapy may require targeting of multiple angiogenic pathways that seem to differ markedly in pGBMs and sGBMs.

Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3beta (GSK3beta) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine-induced Parkinson's dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3beta phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC-PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.

OBJECTIVE

To determine the in vitro effect of nintedanib on primary human lung fibroblasts.

METHODS

Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.

RESULTS

IPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.

CONCLUSIONS

Our data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug's anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β(1) (TGF-β(1) ), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β(1) blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation.

The purpose of this study was to promote fibroblast proliferation and collagen remodeling in flexor tendon repair through sustained delivery of platelet derived growth factor (PDGF-BB). The release kinetics of PDGF-BB from a novel fibrin matrix delivery system was initially evaluated in vitro. After the in vivo degradation rate of the fibrin matrix was determined using fluorescently tagged fibrin, PDGF-BB was delivered to the site of flexor tendon repair in vivo in a canine model. The effect of PDGF-BB on intrasynovial tendon healing was studied using histology-based assays (cell density, proliferation, and type I collagen expression) and by measuring total DNA levels and reducible collagen crosslink levels. The fibrin matrix delivery system provided sustained release of PDGF-BB in vitro at a rate modulated by the ratio of heparin to growth factor. In vivo, the fibrin matrix remained at the repair site for more than 10 days. Delivery of PDGF-BB led to a qualitative increase in cell density, cell proliferation, and type I collagen mRNA expression. PDGF-BB also led to statistically significant increases in total DNA (20% increase at 7 days, 18% increase at 14 days) and reducible collagen crosslinks (30% increase at 7 days). Sustained delivery of growth factors may be achieved using a novel fibrin-based delivery system. PDGF-BB delivery increased cell proliferation and matrix remodeling and thus may accelerate flexor tendon healing.

An in vitro model of VEGF-A-induced angiogenesis was used to generate transcription profiles of human microvascular endothelial cells. Microarray analysis showed increased transcription of genes known to regulate angiogenesis, but also genes that previously have not been firmly associated with angiogenesis such as endocan, pinin, plakophilin, phosphodiesterase 4B and gelsolin. Increased endocan mRNA levels in response to VEGF-A in endothelial cells and in human renal cancer have previously been reported. We now show increased endocan protein levels in VEGF-A treated endothelial cells and in human renal clear cell carcinoma. Increased protein expression was observed both in tumor cells and in a subset of tumor vessels, while expression in normal kidney tissue was low. VEGF-A seemed to be a specific inducer of endocan transcription since FGF-2, PDGF-BB, HGF/SF and EGF did not alter expression levels. Inhibition of PI3K with LY294002 caused a 12-fold increase in endocan transcription suggesting a repressive function of PI3K. In contrast inhibition of Src or MEK, which are signaling pathways activated by VEGF-A, did not influence basal or VEGF-A-induced endocan levels. In conclusion our study shows that, among angiogenic growth factors, VEGF-A is a specific inducer of endocan transcription which is translated into increased protein levels in VEGF-A treated endothelial cells. Increased endocan protein expression in human renal cancer suggests a role in tumor growth.

Binding analyses using 125I-labeled platelet-derived growth factor (PDGF)-AA and PDGF-BB were used to identify a clonal human glioma cell line (U-343 MGa 31L) which expresses the A type but not the B type receptor for PDGF. The glioma cells were devoid of a B type receptor transcript, and immunoprecipitation with an antiserum raised against a B type receptor peptide rendered no signal. Similar analyses using human foreskin fibroblasts, which express both A and B type PDGF receptors, revealed a B type PDGF receptor-specific 5.5-kilobase pair mRNA in a Northern blot experiment, and 160,000 and 180,000 molecular weight components upon immunoprecipitation. A second antiserum, raised against purified porcine PDGF receptor preparations, was reactive with Mr 140,000 and 170,000 components in the U-343 MGa 31L cells, as well as in human fibroblasts. In addition, this antiserum precipitated the Mr 160,000 and 180,000 components from the fibroblasts. Exposure of cells to PDGF-AA, as well as to PDGF-BB, induced an increased rate of degradation of the Mr 170,000 component in the clonal glioma cells and in fibroblasts. The Mr 180,000 component in fibroblasts was degraded only when cells were exposed to PDGF-BB. This allowed the identification of the Mr 170,000 component as the cell surface expressed form of the A type receptor for PDGF. A structural relatedness between the A and B type PDGF receptors was furthermore indicated by similarities in peptide patterns, after limited proteolytic digestion.

We have shown that the G protein-coupled receptor (GPCR) agonists, thrombin and Factor Xa, stimulate smooth muscle cell (SMC) proliferation through transactivation of the EGF receptor (EGFR) or the FGF receptor (FGFR), both of which are tyrosine kinase receptors. In the present study, we investigated whether platelet-derived growth factor (PDGF), a tyrosine kinase receptor agonist, might transactivate another tyrosine kinase receptor to induce SMC proliferation. Because heparin inhibits PDGF-mediated proliferation in human SMCs, we investigated whether the heparin-binding growth factor basic fibroblast growth factor (bFGF) and one of its receptors, FGFR-1, play a role in the response of human arterial SMCs to PDGF-BB. PDGF-BB induced the release of bFGF and sustained phosphorylation of FGFR-1 (30 minutes to 6 hours). A bFGF-neutralizing antibody inhibited PDGF-BB-mediated phosphorylation of FGFR-1, DNA synthesis, and cell proliferation. In the presence of bFGF antibody, PDGF-BB-induced early activation of ERK (0 to 60 minutes) was not affected, whereas late ERK activation (2 to 4 hours) was reduced. When FGFR-1 expression was suppressed using small interfering RNA (siRNA), ERK activation was reduced at late, but not early, time points after PDGF-BB stimulation. Addition of bFGF antibody to cells treated with siRNA to FGFR-1 had no further effect on ERK activation. Our results provide support for a novel mechanism by which PDGF-BB induces the release of bFGF and activation of FGFR-1 followed by the sustained activation of ERK and proliferation of human SMCs.

OBJECTIVE

To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors.

METHODS

The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors.

RESULTS

Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification.

CONCLUSIONS

These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

Vascular pericytes, an important cellular component in the tumor microenvironment, are often associated with tumor vasculatures, and their functions in cancer invasion and metastasis are poorly understood. Here we show that PDGF-BB induces pericyte-fibroblast transition (PFT), which significantly contributes to tumor invasion and metastasis. Gain- and loss-of-function experiments demonstrate that PDGF-BB-PDGFRβ signaling promotes PFT both in vitro and in in vivo tumors. Genome-wide expression analysis indicates that PDGF-BB-activated pericytes acquire mesenchymal progenitor features. Pharmacological inhibition and genetic deletion of PDGFRβ ablate the PDGF-BB-induced PFT. Genetic tracing of pericytes with two independent mouse strains, TN-AP-CreERT2:R26R-tdTomato and NG2-CreERT2:R26R-tdTomato, shows that PFT cells gain stromal fibroblast and myofibroblast markers in tumors. Importantly, coimplantation of PFT cells with less-invasive tumor cells in mice markedly promotes tumor dissemination and invasion, leading to an increased number of circulating tumor cells and metastasis. Our findings reveal a mechanism of vascular pericytes in PDGF-BB-promoted cancer invasion and metastasis by inducing PFT, and thus targeting PFT may offer a new treatment option of cancer metastasis.

The purpose of this study was to evaluate the vasoformative response of isolated vascular explants to a variety of growth factors that have been shown to stimulate angiogenesis. Rings of rat aorta were cultured in collagen gels under serum-free conditions in the presence or absence of vascular endothelial growth factor (VEGF), natural platelet-derived growth factor (PDGF), PDGF-AA, PDGF-BB, insulin-like growth factor-1 (IGF-1), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), interleukin-1 alpha (IL-1 alpha), or hepatocyte growth factor (HGF). The angiogenic response of the rat aorta was stimulated by VEGF, PDGF, PDGF-AA, PDGF-BB, and IGF-1. Maximum stimulatory effects were obtained with VEGF and PDGF-BB. By contrast, TGF-beta 1 and IL-1 alpha had inhibitory activity. No significant effects were observed with TGF-alpha, EGF, or HGF. The vascular outgrowth of VEGF-stimulated cultures was primarily composed of microvessels, whereas that of PDGF- and IGF-1-stimulated cultures contained an increased number of fibroblast-like cells. The inability of TGF-alpha, TGF-beta 1, IL-1 alpha, EGF, and HGF to stimulate rat aortic angiogenesis in serum-free culture suggests that either these factors require the mediatory activity of accessory cells that are not present in the rat aorta model or that blood vessels are heterogeneous in their capacity to respond to different angiogenic factors.

Platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors play instrumental roles in embryonic organogenesis and diseases of adult organs. In particular, platelet-derived growth factor receptor-alpha (PDGFRα) is expressed by multipotent cardiovascular progenitors in mouse and human embryonic stem cell systems. Although cardiac PDGFRα expression has been studied in multiple species, little is known about its expression in the human heart. Using immunofluorescence, we analyzed PDGFRα expression in both human fetal and diseased adult hearts, finding strong expression in the interstitial cells of the epicardium, myocardium, and endocardium, as well as the coronary smooth muscle. Only rare endothelial cells and cardiomyocytes expressed PDGFRα. This pattern was consistent for both the fetal and adult diseased hearts, although more PDGFRα+ cardiomyocytes were noted in the latter. In vitro differentiation assays were then performed on the PDGFRα+ cell fraction isolated from the cardiomyocyte-depleted human fetal hearts. Protocols previously reported to direct differentiation to a cardiomyocyte (5-azacytidine), smooth muscle (PDGF-BB), or endothelial cell fates (vascular endothelial growth factor [VEGF]) were used. Although no significant cardiomyocyte differentiation was observed, PDGFRα+ cells generated significant numbers of smooth muscle cells (smooth muscle-α-actin+ and smooth muscle myosin+) and endothelial cells (CD31+). These data suggest that a subfraction of the cardiac PDGFRα+ populations are progenitors contributing predominantly to the vascular and mesenchymal compartments of the human heart. It may be possible to control the fate of these progenitors to promote vascularization or limit fibrosis in the injured heart.

BACKGROUND

The development of platelet-rich fibrin (PRF) drastically simplified the preparation procedure of platelet-concentrated biomaterials, such as platelet-rich plasma (PRP), and facilitated their clinical application. PRF's clinical effectiveness has often been demonstrated in pre-clinical and clinical studies; however, it is still controversial whether growth factors are significantly concentrated in PRF preparations to facilitate wound healing and tissue regeneration. To address this matter, we performed a comparative study of growth factor contents in PRP and its derivatives, such as advanced PRF (A-PRF) and concentrated growth factors (CGF).

METHODS

PRP and its derivatives were prepared from the same peripheral blood samples collected from healthy donors. A-PRF and CGF preparations were homogenized and centrifuged to produce extracts. Platelet and white blood cell counts in A-PRF and CGF preparations were determined by subtracting those counts in red blood cell fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts of whole blood samples. Concentrations of growth factors (TGF-β1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1β, IL-6) were determined using ELISA kits.

RESULTS

Compared to PRP preparations, both A-PRF and CGF extracts contained compatible or higher levels of platelets and platelet-derived growth factors. In a cell proliferation assay, both A-PRF and CGF extracts significantly stimulated the proliferation of human periosteal cells without significant reduction at higher doses.

CONCLUSIONS

These data clearly demonstrate that both A-PRF and CGF preparations contain significant amounts of growth factors capable of stimulating periosteal cell proliferation, suggesting that A-PRF and CGF preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application.

We have investigated the use of natural and synthetic collagenous matrices as carriers of exogenous growth factors. A bladder acellular matrix (BAM) was processed from rat bladder and compared with sponge matrix of porcine type 1 collagen. The lyophilized matrices were rehydrated by the aqueous solutions of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), insulin like growth factor-1 (IGF-1) and heparin binding epidermal growth factor-like growth factor (HB-EGF), to obtain the matrix incorporating each growth factor. The rehydration method enabled the growth factor protein to distribute into the matrix homogeneously. In vivo release test in the mouse subcutis revealed that, the property of BAM for growth factor release was similar to that of collagen sponge. Among the growth factors examined, bFGF release was the most sustained, followed by HGF and PDGF-BB. bFGF released from the two matrices showed similar in vivo angiogenic activity at the mouse subcutis in a dose-dependent manner. These findings demonstrate that the collagenous matrices function as release carriers of growth factors. This feature is promising to create a scaffold, which has a nature to control the tissue regeneration actively.

OBJECTIVE

The aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion.

METHODS

Three groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF(165) (VEGF), or alginate containing VEGF(165) combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points.

RESULTS

In the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles.

CONCLUSIONS

Sequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.

Tumor cells recruit vascular endothelial cells and circulating endothelial progenitor cells to form new vessels to support their own growth and metastasis. VEGF, PDGF-BB and FGF-2 are three major pro-angiogenic factors and applied to promote angiogenesis. In this research, we demonstrated that anlotinib, a potent multi-tyrosine kinases inhibitor (TKI), showed a significant inhibitory effect on VEGF/PDGF-BB/FGF-2-induced angiogenesis in vitro and in vivo. Wound healing assay, chamber directional migration assay and tube formation assay indicated that anlotinib inhibited VEGF/PDGF-BB/FGF-2-induced cell migration and formation of capillary-like tubes in endothelial cells. Furthermore, anlotinib suppressed blood vessels sprout and microvessel density in rat aortic ring assay and chicken chorioallantoic membrane (CAM) assay. Importantly, according to our study, the anti-angiogenic effect of anlotinib is superior to sunitinib, sorafenib and nintedanib, which are three main anti-angiogenesis drugs in clinic. Mechanistically, anlotinib inhibits the activation of VEGFR2, PDGFRβ and FGFR1 as well their common downstream ERK signaling. Therefore, anlotinib is a potential agent to inhibit angiogenesis and be applied to tumor therapy.

We report an electrochemical, aptamer-based (E-AB) sensor for the detection of platelet-derived growth factor (PDGF) directly in blood serum. The E-AB approach employs alternating current voltammetry to monitor target-induced folding in a methylene blue-modified, PDGF-binding aptamer. The sensor is sensitive, highly selective, and essentially reagentless: we readily detect the BB variant of PDGF at 1 nM directly in undiluted, unmodified blood serum and at 50 pM (1.25 ng/mL) in serum-diluted 2-fold with aqueous buffer. The sensitivity and selectivity achieved by this sensor match or significantly exceed those of the best analogous optical approaches. For example, the detection limit attained in 50% serum is achieved against a >25 million-fold excess of contaminating blood proteins and represents a 4 order of magnitude improvement over the most sensitive optical PDGF aptasensor reported to date. Moreover, the E-AB sensor combines these promising attributes in a platform that is reusable, label-free, and electronic. Given these advantages, E-AB sensors appear well suited for implementation in portable microdevices directed at the direct detection of proteins and small molecules in complex, largely unprocessed clinical samples.