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PubMed2006

Nano-fibrous scaffold for controlled delivery of recombinant human PDGF-BB.

The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.

Becaplermin↗
PubMed2006

PDGF isoforms induce and maintain anagen phase of murine hair follicles.

BACKGROUND: It is known that platelet-derived growth factor (PDGF) receptors are expressed in hair follicle (HF) epithelium. OBJECTIVES: The aim of the present study was to clarify the effects of PDGF-AA and -BB on the cyclic growth of HFs. METHODS: PDGF-AA or -BB was injected into the dorsal skin of C3H mice during the second telogen phase once daily for five consecutive days, or PDGF-AA or -BB dissolved in hyaluronic acid was injected only once. In order to confirm the effects of different PDGF isoforms, anti-PDGF-AA antibody or anti-PDGF-BB antibody was injected just after each injection of PDGF-AA or -BB. In addition, anti-PDGF antibodies were injected into the skin of C3H mice during the second anagen phase once daily for 5 days. We studied expression of signaling molecules in the skin where anagen phase had been induced by PDGF injection by real-time RT-PCR. RESULTS: Both PDGF-AA and -BB injection experiments immediately induced the anagen phase of the hair growth cycle at the injection sites. The induction of anagen was interfered by anti-PDGF antibody treatment. Real-time RT-PCR using extracted RNA from the PDGF injected sites of skin samples showed upregulated expression of HF differentiation-related key signaling molecules, Sonic hedgehog (Shh), Lef-1 and Wnt5a. CONCLUSIONS: These results indicate that both PDGF-AA and -BB are involved in the induction and maintenance of the anagen phase in the mouse hair cycle. Local application of PDGF-AA and -BB might therefore prove to be an effective treatment option for alopecia associated with early catagen induction and elongated telogen phase.

Animals↗
PubMedDate not supplied

Differential effects of PDGF-BB on matrix metalloproteases and cytokine release in fibroblasts of Type 2 diabetic patients and normal controls in vitro.

AIMS/HYPOTHESIS: The complex process of wound healing is regulated by various growth factors. The systemic character of diabetes mellitus favors the chronification of diabetic wounds. In this study, the in vitro effects of platelet-derived growth factor (PDGF)-BB on the expression of cytokines and matrix metalloproteases (MMPs) in fibroblasts of Type 2 diabetic patients and healthy controls were investigated. METHODS: We studied six Type 2 diabetic patients (mean Hba1(c)=7.5%) and six healthy controls. For proliferation studies, cultivated fibroblasts, prepared from biopsies taken from the thigh, were stimulated with different concentrations of PDGF. After 48 h, the expression of MMPs and cytokines was measured. We analysed the mRNA expression by RT-PCR (TaqMan), tissue protein levels by zymography, and cell supernatant levels by ELISA. RESULTS: Levels of MMP-mRNA were elevated in diabetic fibroblasts compared with healthy controls. At baseline, MMP-2 protein levels were significantly increased in the fibroblast of diabetic patients (P=.019). For MMP-9, a trend towards higher levels (P=.3) was found. After incubation with PDGF, a significant reduction of MMP-9 (P=.01) and MMP-13 (P=.04) was found. Analysis of cytokine release in cell culture supernatant showed elevated levels of interleukin (IL)-8 at baseline conditions. MMP-1 and MMP-2 levels in the supernatant were concentration-dependently reduced. CONCLUSIONS: This study, for the first time, demonstrates elevated MMPs in cultivated fibroblasts (derived from intact skin and not from an open wound) of diabetic patients compared with healthy controls under in vitro conditions. Therefore, our data support the hypothesis of alterations of wound healing in diabetic patients on the cellular level, reflecting the systemic character of the disease.

Becaplermin↗
PubMed2004

Herb medicine Inchin-ko-to (TJ-135) regulates PDGF-BB-dependent signaling pathways of hepatic stellate cells in primary culture and attenuates development of liver fibrosis induced by thioacetamide administration in rats.

BACKGROUND/AIMS: We studied the effect of Inchin-ko-to (TJ-135), a herb medicine that has been clinically used for liver cirrhosis in Japan, on liver fibrosis in a rat model and on the function of stellate cells. METHODS: Rat liver fibrosis was generated by thioacetamide (TAA) administration. DNA synthesis was assessed by 5-bromo-2'-deoxyuridine incorporation assay. Protein expression was analysed by western blotting. Collagen and fibronectin mRNA expression were analysed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: TJ-135 improved liver fibrosis induced in rats by TAA administration. TJ-135 reduced collagen deposition and the expression of smooth muscle alpha-actin in fibrotic liver tissues and decreased the serum level of hyaluronic acid. In primary-cultured stellate cells, TJ-135 suppressed DNA synthesis and the expression of collagen alpha 1(I), collagen III, and fibronectin mRNAs. It hampered DNA synthesis and migration of PDGF-BB-stimulated stellate cells through inhibiting phosphorylation of PDGF receptor-beta and down-stream signaling pathways. Among TJ-135 components, 3-methyl-1,6,8-trihydroxyanthraquinone (emodin) derived from Rhei rhizoma was found to be the most active molecule. CONCLUSIONS: TJ-135 and emodin regulate PDGF-dependent events in stellate cells and attenuate the development of liver fibrosis. Their clinical use may be beneficial for the therapy of human liver fibrosis.

Actins↗
PubMed2006

Single variable domain antibody as a versatile building block for the construction of IgG-like bispecific antibodies.

Bispecific antibodies (BsAb) have been traditionally utilized to redirect cytotoxic effector cells and agents to kill tumor cells expressing the target antigens. Recently a new concept is emerging to develop BsAb that simultaneously block the functions of two tumor-associated targets, eg., growth factor receptors, for enhanced antitumor efficacies. Broad clinical applications of BsAb have been, and still are, significantly hampered by the difficulty in producing the materials in sufficient quantity and quality by traditional approaches. Here we describe a recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, using a single variable domain (sVD) antibody as a versatile building block. In this method, a sVD of a defined specificity is genetically fused to either the N-terminus of the light chain or the C-terminus of the heavy chain of a functional IgG antibody of a different specificity. A model BsAb was constructed using a sVD to mouse platelet derived growth factor receptor alpha and a conventional IgG antibody to mouse platelet derived growth factor receptor beta. The BsAb were expressed in mammalian cells and purified to homogeneity by a one-step Protein A affinity chromatography. Further, the BsAb retained the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. Importantly, the BsAb inhibited the activation of both its target receptors in tumor cells stimulated by both platelet derived growth factor AA and BB, whereas the parent monospecific antibody only inhibited the activation of a single receptor stimulated by its cognate ligand. This format of BsAb should be readily applicable to the production of other BsAb recognizing any pairs of antigens.

Animals↗
PubMedDate not supplied

Expression of hyaluronan synthase in intraocular proliferative diseases: regulation of expression in human vascular endothelial cells by transforming growth factor-beta.

PURPOSE: To investigate the role of hyaluronan (HA) and elucidate the mechanisms that regulate the expression of hyaluronan-synthesizing enzymes in vascular endothelial cells (VECs) in intraocular proliferative diseases. METHODS: Cultured VECs were used. Hyaluronan synthase (HAS) expression was determined on the mRNA products obtained by reverse transcription polymerase chain reaction (RT-PCR). The effect of transforming growth factor-beta(1)(TGF-beta(1)) and/or platelet-derived growth factor-BB (PDGF-BB) on HAS expression was examined by quantitative RT-PCR and Western blot analysis. HAS expression in intraocular proliferative membranes was observed by immunohistochemistry. RESULTS: Cultured VECs expressed the three HAS isoforms. Stimulation of VECs with TGF-beta(1) induced a marked increase in the expression level of HAS2 mRNA and protein. The stimulatory effect of PDGF-BB was less potent. A synergistic or additive effect between TGF-beta(1) and PDGF-BB-induced HA synthesis was not observed. Furthermore, HAS1 and HAS2 exhibited differential expression in VECs and non-VECs populating intraocular proliferative membranes. CONCLUSIONS: The expression of each HAS isoform is regulated differently by growth factors and cytokines in VECs. Importantly, HA-synthesizing enzymes were expressed in cells populating proliferative membranes obtained from eyes of patients with proliferative vitreoretinal diseases, and thus may be key molecules in the events that control progression of the proliferative diseases.

Becaplermin↗
PubMed2005

Upregulation of vascular growth factors in multiple sclerosis: correlation with MRI findings.

Vascular permeability changes precede the development of demyelinating lesions in multiple sclerosis (MS), and vessel wall thickening and capillary proliferation are frequently seen in autopsied MS lesions. Although vascular growth factors are critical for inducing such vascular changes, their involvement in MS has not been extensively studied. Thus, we examined the involvement of various vascular growth factors in MS according to their clinical phase and subtype. We measured serum levels of vascular endothelial growth factor (VEGF), acidic and basic fibroblast growth factors (FGF) and platelet-derived growth factors (PDGFs)-AA, -AB and -BB in 50 patients with MS (27 opticospinal MS and 23 conventional MS patients) and 33 healthy controls using sandwich enzyme immunoassays. Correlations between growth factor changes and brain and spinal cord MRI findings were then analyzed. Serum VEGF concentrations were significantly higher in MS patients in relapse than in controls (p = 0.0495) and in MS patients in remission (p = 0.0003), irrespective of clinical subtype. Basic FGF was significantly increased in conventional MS patients, but not opticospinal MS patients compared with controls (p = 0.0291), irrespective of clinical phase. VEGF at relapse showed a significant positive correlation with the length of spinal cord lesions on MRI (r = 0.506, p = 0.0319). The results suggest that an increase in serum VEGF concentration might be involved in MS relapse and the formation of longitudinally extensive spinal cord lesions.

Adult↗
PubMed2004

Age related changes in human articular chondrocyte yield, proliferation and post-expansion chondrogenic capacity.

OBJECTIVE: We investigated how aging effects human chondrocyte yield, proliferation, post-expansion chondrogenic capacity, and response to specific growth factors supplemented during expansion. METHODS: Fifty-two samples of human articular cartilage were harvested from cadavers 20 to 91 years old and grouped into age decades. Cell yields were normalised to tissue wet weight. Cell proliferation rates were calculated during expansion in medium without (CTR) or with TGF beta 1, FGF-2 and PDGF-BB (TFP). Chondrogenic capacity of CTR- and TFP-expanded cells was assessed by cultivation as 3D pellets in a defined serum-free medium, followed by histological, immunohistochemical, biochemical and real-time RT-PCR analyses. RESULTS: Cell yields were similar in donors up to 40 years of age and significantly lower (1.8-fold) in older donors. Cell proliferation rates in CTR medium significantly decreased after 30 years of age and remained similar in older donors. In the presence of TFP, proliferation rates were higher (up to 3.7-fold) in all age groups and decreased only slightly with age. The glycosaminoglycan (GAG) content of pellets obtained from CTR-expanded cells was not correlated with age. Pellets from TFP-expanded cells reproducibly contained more GAG (up to 3.2-fold) than those from CTR-expanded cells only if donors were younger than 40. Safranin O staining intensity and collagen type II expression and accumulation were consistent with GAG contents. CONCLUSION: Medium supplementation with the growth factor combination TFP during chondrocyte expansion supports higher proliferation rates at any age and higher post-expansion chondrogenic capacity in donors up to 40 years. These findings may be relevant for chondrocyte-based cartilage repair procedures.

Adult↗
PubMed2005

Calcitriol inhibits growth response to Platelet-Derived Growth Factor-BB in human prostate cells.

Calcitriol, a hormonal form of Vitamin D, regulates growth of normal and cancer cells of various origins by modulation of peptide growth factors signaling. Platelet-Derived Growth Factor (PDGF) signaling pathway is involved in prostate cancer progression. We studied the expression of PDGF receptors in human prostate primary stromal cells and cancer epithelial cell lines and growth response to PDGF-BB isoform. We found that the expression of PDGF receptors and PDGF-BB-mediated cell growth are regulated by calcitriol in prostate cells. Quantitative RT-PCR analysis revealed a lower level of mRNA for PDGF receptors in LNCaP and PC-3 cells than in primary stromal cells. Western blotting showed a high amount of PDGFRalpha and beta proteins in primary stromal cells that could not be detected in LNCaP, which may explain the resistance of LNCaP cells to growth-promoting effect of PDGF-BB. Addition of Epidermal Growth Factor (EGF) to the culture medium induces the expression of PDGFRbeta and restores responsiveness of LNCaP to PDGF-BB to some extent. Calcitriol down-regulates PDGFRbeta expression and negatively regulates PDGF-mediated cell growth. Calcitriol does not affect PDGFRalpha and PDGF-B mRNA expression. We suggest that inhibition of PDGFRbeta expression by calcitriol might reduce responsiveness of prostate cells to mitogenic action of PDGF-BB.

Base Sequence↗
PubMed2004

Interleukin-1beta inhibits PDGF-BB-induced migration by cooperating with PDGF-BB to induce cyclooxygenase-2 expression in baboon aortic smooth muscle cells.

OBJECTIVE: Smooth muscle cell (SMC) migration from the media into the intima is pivotal for intimal formation after vascular injury. Platelet-derived growth factor (PDGF)-BB is a potent chemoattractant for SMCs in vitro and in vivo. We investigated whether interleukin (IL)-1beta affects migration in response to PDGF-BB. Our data suggest that IL-1beta is inhibitory and that this effect is mediated by cyclooxygenase (COX)-2. We further addressed the role of the mitogen-activated protein kinase p38, which is activated by PDGF-BB and by IL-1beta. METHODS: Baboon aortic SMCs were prepared with the explant method. Migration was measured in a Boyden chamber assay through filters coated with monomeric collagen. COX2 expression and phosphorylation of p38 MAPK were analyzed by Western blotting. RESULTS: PDGF-BB (10 ng/mL) stimulates migration 3.8-fold, and IL-1beta (0.1 ng/mL) reduces this response by 40%. The inhibitory effect of IL-1beta is abolished by the COX inhibitor, indomethacin (10 micromol/L), the specific COX2 inhibitor, NS398 (10 micromol/L), and the p38 MAPK inhibitor SB203580 (3 micromol/L). We found that IL-1beta and PDGF-BB synergize to stimulate COX2 expression. We further demonstrated that p38 MAPK is activated by IL-1beta and PDGF with different kinetics and that p38 MAPK is required for maximal COX2 expression in response to IL-1beta plus PDGF-BB. CONCLUSION: IL-1beta inhibits PDGF-BB-induced migration by cooperating with PDGF-BB to induce COX2 through activation of p38 MAPK. Whether this effect of IL-1beta modulates intimal growth after vascular injury remains to be elucidated.

Animals↗
PubMed2005

Shear stress-stimulated endothelial cells induce smooth muscle cell chemotaxis via platelet-derived growth factor-BB and interleukin-1alpha.

OBJECTIVE: Vascular smooth muscle cell (SMC) migration is critical to the development of atherosclerosis and neointimal hyperplasia. Hemodynamic forces such as shear stress and cyclic strain stimulate endothelial cell signal-transduction pathways, resulting in the secretion of several factors, including SMC chemoattractants such as platelet-derived growth factor (PDGF). We hypothesized that mechanical forces stimulate endothelial cells to secrete SMC chemoattractants to induce migration via the mitogen-activated protein kinase (MAPK) pathway. METHODS: Bovine aortic endothelial cells were exposed to shear stress, cyclic strain, or static conditions for 16 hours. The resulting conditioned medium was used as a SMC chemoattractant in a Boyden chamber. Activation of SMC extracellular signal-regulated protein kinase 1/2 (ERK1/2) was assessed by Western blot analysis. Pathways were inhibited with anti-PDGF-BB or anti-interleukin-1alpha (IL-1alpha) antibodies, or the ERK1/2 upstream pathway inhibitor PD98059. RESULTS: Conditioned medium from endothelial cells exposed to shear stress corresponding to arterial levels of shear stress stimulated SMC migration but lower levels of shear stress or cyclic strain did not. Both PDGF-BB and IL-1alpha were secreted into the conditioned medium by endothelial cells stimulated with shear stress. Both PDGF-BB and IL-1alpha stimulated SMC chemotaxis but were not synergistic, and both stimulated SMC ERK1/2 phosphorylation. Inhibition of PDGF-BB or IL-1alpha inhibited SMC chemotaxis and ERK1/2 phosphorylation. CONCLUSION: Shear stress stimulates endothelial cells to secrete several SMC chemoattractants, including PDGF-BB and IL-1alpha; both PDGF-BB and IL-1alpha stimulate SMC chemotaxis via the ERK1/2 signal-transduction pathway. These results suggest that the response to vascular injury may have a common pathway amenable to pharmacologic manipulation. CLINICAL RELEVANCE: One difficulty in the pharmacologic treatment of atherosclerosis or neointimal hyperplasia leading to restenosis is the multiplicity of activated pathways and thus potential treatment targets. This study demonstrates that shear stress, a hemodynamic force that may be a biologically relevant stimulus to induce vascular pathology, stimulates endothelial cells to secrete PDGF-BB and IL-1alpha. Both of these mediators stimulate the SMC ERK1/2 pathway to induce migration, a critical event in the pathogenesis of atherosclerosis and neointimal hyperplasia. Therefore, this study suggests a relevant common target pathway in SMC that is amenable to manipulation for clinical treatment.

Animals↗
PubMed2005

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells.

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.

Androstadienes↗
PubMed2007

Improved growth factor directed vascularization into fibrin constructs through inclusion of additional extracellular molecules.

Using the chick chorioallantoic membrane assay (CAM) and a novel histological technique, we investigated the ability of blood vessels to directly invade fibrin-based scaffolds. In our initial experiments utilizing vascular endothelial growth factor (VEGF(165)), we found no direct invasion. Instead, the fibrin was completely degraded and replaced with highly vascularized new tissue. Addition of fibroblast growth factor-2 (FGF-2), bone morphogenic protein-2 (BMP-2), or platelet-derived growth factor-BB (PDGF-BB) to the fibrin construct also did not result in construct vascularization. Because natural and regenerating tissues exhibit complex extracellular matrices (ECMs), we hypothesized that a more complex scaffold may improve blood vessel invasion. Addition of fibronectin, hyaluronic acid, and collagen type I within 20 mg/mL fibrin constructs resulted in no significant improvement. However, the same additive concentrations within 10 mg/mL fibrin constructs resulted in dramatic improvements, specifically with hyaluronic acid. Overall, we believe that these results indicate the importance of structural and functional cues of not only in the initial scaffold but also as the construct is degraded and remodeled. Furthermore, the CAM assay may represent a useful model for understanding ECM interactions as well as for screening and designing tissue-engineered scaffolds.

Allantois↗
PubMedDate not supplied

Platelet-derived growth factor-BB pretreatment attenuates excitotoxic death in cultured hippocampal neurons.

Neuronal excitotoxic death results from excess stimulation by elevated levels of extracellular glutamate acting on N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. While excitotoxicity is typically attenuated by using glutamate receptor antagonists, we report here that neuronal deaths induced directly by brief exposures to glutamate or NMDA were both attenuated by preincubation with platelet-derived growth factor-BB (PDGF-BB). The neuroprotection was concentration and time dependent; preincubation for at least 24 h with a minimum of 10 ng/mL of PDGF-BB was required for maximal neuroprotective effect. The NMDA receptor antagonist MK-801 also afforded partial protection, and when MK-801 was used with PDGF-BB, neuronal survival was comparable to that of untreated controls. When protection of inhibitory and excitatory neurons by PDGF treatment was compared, the excitatory neurons appeared to be selectively protected. The present results demonstrate that PDGF pretreatment can protect neurons from direct glutamate-induced excitotoxicity in vitro and suggests that PDGF might possibly function as a neuroprotective agent in potential therapeutic applications.

Animals↗
PubMed2005

Expression and purification of recombinant vascular endothelial-statin.

We have recently discovered VE-statin (egfl7), a novel protein specifically expressed by endothelial cells during mouse development and in the adult. VE-statin is expected to play an essential role during the formation of new blood vessels. Here, we have expressed the coding sequence for VE-statin in bacteria and designed a protocol for the production and purification of large amounts of the protein. The protocol is based on an affinity purification step followed by a strong cation-exchange chromatography and a refolding procedure adapted to epidermal growth factor domains and also to the high tendency of the protein to precipitate in concentrated solutions. The purified protein is pure as assessed by SDS-PAGE, immunoblot and mass spectrometry, and is biologically active as a repressor of human aortic smooth muscle cell migration induced by platelet-derived growth factor-BB.

Amino Acid Sequence↗
PubMed2005

Atrial natriuretic peptide in hypoxia.

A growing number of mammalian genes whose expression is inducible by hypoxia have been identified. Among them, atrial natriuretic peptide (ANP) synthesis and secretion is increased during hypoxic exposure and plays an important role in the normal adaptation to hypoxia and in the pathogenesis of cardiopulmonary diseases, including chronic hypoxia-induced pulmonary hypertension and vascular remodeling, and right ventricular hypertrophy and right heart failure. This review discusses the roles of ANP and its receptors in hypoxia-induced pulmonary hypertension. We and other investigators have demonstrated that ANP gene expression is enhanced by exposure to hypoxia and that the ANP so generated protects against the development of hypoxic pulmonary hypertension. Results also show that hypoxia directly stimulates ANP gene expression and ANP release in cardiac myocytes in vitro. Several cis-responsive elements of the ANP promoter are involved in the response to changes in oxygen tension. Further, the ANP clearance receptor NPR-C, but not the biological active NPR-A and NPR-B receptors, is downregulated in hypoxia adapted lung. Hypoxia-sensitive tyrosine kinase receptor-associated growth factors, including fibroblast growth factor (FGF) and platelet derived growth factor (PDGF)-BB, but not hypoxia per se, inhibit NPR-C gene expression in pulmonary arterial smooth muscle cells in vitro. The reductions in NPR-C in the hypoxic lung retard the clearance of ANP and allow more ANP to bind to biological active NPR-A and NPR-B in the pulmonary circulation, relaxing preconstricted pulmonary vessels, reducing pulmonary arterial pressure, and attenuating the development of hypoxia-induced pulmonary hypertension and vascular remodeling.

Animals↗
PubMed2005

Growth-promoting effect of platelet-derived growth factor on rat cardiac myocytes.

Platelet-derived growth factor (PDGF) is a dimeric molecule consisting of disulfide-bonded A- and B-polypeptide chains. Homodimeric (PDGF-AA, PDGF-BB) as well as heterodimeric (PDGF-AB) isoforms exert their effects on target cells by binding with different specificities to two structurally related protein tyrosine kinase receptors, denoted alpha- and beta-receptors. PDGF stimulates growth in various cell types, but little is known about its effect on mammalian cardiomyocytes. Therefore, growth-promoting effect of PDGF on rat cardiomyocytes was investigated. Primary culture of neonatal rat ventricular myocytes was prepared and cellular growth was estimated by [3H]-leucine incorporation assay. Tyrosine-phosphorylated PDGF-beta receptor of cardiomyocytes was determined by immunoblotting analysis after immunoprecipitation. PDGF-beta receptor, extracellular signal-regulated kinase (ERK) 1/2 and phosphorylated ERK1/2 of cardiomyocytes were measured by immunoblotting analysis. [3H]-leucine incorporation into the cultured myocytes was increased in a time- and dose-dependent manner after PDGF-BB stimulation. Phosphorylation of PDGF-beta receptor and ERK1/2 in cardiomyocytes was increased after short-term stimulation of PDGF-BB. Protein expression of PDGF-beta receptor and ERK1/2 was increased after long-term stimulation of PDGF-BB. [(3)H]-leucine incorporation into the cultured myocytes induced by PDGF-BB was partly blocked by mitogen-activated ERK-activating kinase (MEK) inhibitor PD98059, phospholipase C (PLC) inhibitor U73122, and protein kinase C (PKC) inhibitor staurosporin aglycone, respectively. Therefore, PDGF beta receptor, ERK1/2, PLC and PKC are involved in the signal transduction of PDGF-induced growth response of rat cardiac myocytes.

Animals↗
PubMed2005

Normal human fibroblasts enable melanoma cells to induce angiogenesis in type I collagen.

BACKGROUND: We previously reported that fibroblasts induce human microvascular endothelial cells (HMVECs) to differentiate from monolayer to capillarylike morphology. We now test the hypothesis that fibroblasts modulate angiogenesis in melanoma cells. METHODS: We tested 12 human melanoma lines (2 radial growth phase (RGP), 3 vertical growth phase (VGP), and 7 metastatic (MM)) for ability to induce HMVECs to invade/migrate into collagen and form capillarylike networks. HMVEC monolayers were overlaid with 3-dimensional collagen gels embedded with melanoma cells alone (M), fibroblasts alone (F), or a 1:1 mixture of the 2 cells (M+F). After 5 days, gels were removed, fixed, and HMVEC networks were quantified by von Willebrand's factor (vWF) immunofluorescence. The influence of soluble factors on HMVEC invasion/migration into collagen was assessed with the use of acellular 3-D collagen gels overlaid on HMVEC monolayers, cultured with conditioned media (CM) derived from monolayers of M, F, or M+F. Angiogenic growth factors involved in the observed invasion/migration were identified with the use of a RayBio Cytokine Antibody Array (RayBiotech, Norcross, Ga). RESULTS: Cell line-specific variability in melanoma-supported angiogenesis was observed only when in combination with fibroblasts (analysis of variance [ANOVA], P < .01). Melanoma plus fibroblasts uniformly resulted in a significantly higher angiogenic response than melanoma alone (P < .05). One vertical growth phase and one metastatic melanoma line, while weakly angiogenic alone, induced significantly higher angiogenesis than either fibroblast or melanoma alone (P < .05) when combined with fibroblasts. CM from M or M+F induced significantly less HMVEC invasion/migration into collagen than CM from fibroblasts alone. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 were identified as significantly elevated in the media derived from M+F cultures, compared with either cell type alone. CONCLUSION: To our knowledge, this is the first report demonstrating that melanoma-supported angiogenesis in collagen is more significantly influenced by normal skin-derived fibroblasts than by the intrinsic biology of the melanoma cell type. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 are implicated as potential paracrine factors regulating this observed effect.

Angiogenic Proteins↗