Recent studies reveal conflicting results on the change of solute transfer with time on continuous ambulatory peritoneal dialysis (CAP<em>D</em>) and recurrent peritonitis. Herein, we performed a cross-sectional study of 76 patients on CAP<em>D</em> to examine their peritoneal permeability by measuring the dialysate to serum ratio of creatinine (<em>D</em>/P) and the mass transfer area coefficients of creatinine (MTACCr) or glucose (MTACGlu). Transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor (<em>PDGF</em>), and epidermal growth factor (EGF) were measured in the dialysate by ELISA. TGF-beta1 mRNA in peritoneal macrophages were determined by a quantitative polymerase chain reaction. We failed to observe any correlation between the duration on dialysis and the peritoneal permeability in those patients with no previous peritonitis. Frequency of peritonitis episode did not affect the MTACCr, MTACGlu, or <em>D</em>/P. The MTACCr correlated well with MTACGlu (r = 0.78, p = 0. 001) and with <em>D</em>/P (r = 0.98, p < 0.0001). No inverse correlation was demonstrated between dialysate <em>PDGF</em> or EGF and the peritoneal permeability. A positive correlation was demonstrated between the dialysate TGF-beta1 and MTACCr, MTACGlu or <em>D</em>/P (r = 0.64, 0.54, and 0.64 respectively, p < 0.001). The dialysate TGF-beta1 levels in patients with low <em>D</em>/P (</=0.5) were only half of that in patients with normal or high <em>D</em>/P (p = 0.0002). The dialysate levels of TGF-beta1 did not correlate with <em>PDGF</em> or EGF. These findings raise the possibility that, other than diffusion across the peritoneal membrane from circulation, there could also be an intrinsic production of TGF-beta1 by peritoneal cells in these CAP<em>D</em> patients. Our findings raise the speculation that TGF-beta1 in dialysate from stable CAP<em>D</em> patients may exert an inhibitory action to fibroblast. Such action of TGF-beta1 could reduce the risk of peritoneal sclerosis and hence, maintains a satisfactory peritoneal permeability to small solutes.

Chemotaxis of blood monocytes into the vessel wall together with the change of the relative content of the extracellular matrix (ECM) proteins at sites of predilection is an early cellular marker of atherogenesis. To examine the influence of ECM proteins on secretion of chemoattractants by endothelial cells (EC), porcine EC were seeded on gelatin (G), fibronectin (Fn) and fibrinogen (Fg). After 24 h cells seeded on G and Fn showed the histiotypic 'cobblestone'-morphology whereas cells seeded on Fg did not. Chemotactic activity for monocytes in supernatants from cells seeded on Fg was more than two-fold higher compared with G and was independent of soluble Fn or Fg in the supernatant. Quantification of monocyte chemoattracting protein-1, PDGF-AB and IL-8 in EC supernatants showed that Fg led to a significant increase in secretion of all three proteins compared with cells cultured on G. Preincubation of porcine EC with the tripeptide arginine-glycine-aspartic acid, as inhibitor of binding of Fg to integrin receptors, but not with the control tripeptide arginine-glycine-glutamic acid showed a decrease in chemotactic activity for cells cultured on Fg but not on Fn or G. Inhibition of protein kinase C (PKC) activity in EC by GF109203 resulted in a decrease of fibrinogen-induced chemotactic activity. Also the tyrosine-kinase inhibitor herbimycin inhibited fibrinogen mediated secretion of chemokines. The role of the PKC pathway for matrix mediated signal transduction is further corroborated by Fg-dependent induction of the PKC isoform delta. These data indicate an integrin-dependent signal transduction pathway leading to induction of chemotactic activity by the ECM protein fibrinogen. This mechanism may contribute to induction of chemokines in early atherosclerotic lesions.

The mechanism by which the bradykinin B1 receptor (B1R) inhibits platelet-derived growth factor (PDGF)-stimulated proliferation was investigated in cultured rat mesenteric arterial smooth muscle cells. The B1R agonist des-Arg9-bradykinin (DABK) was found to inhibit PDGF-mediated activation of the cyclin E-cyclin-dependent kinase 2 (Cdk2) complex and to prevent hyperphosphorylation of retinoblastoma protein. DABK did not inhibit upregulation of cyclin E expression but increased expression of the Cdk2 inhibitor p27Kip1 and the association of p27Kip1 with the cyclin E-Cdk2 complex. In addition, DABK inhibited the PDGF-stimulated expression of cyclin D that would otherwise siphon p27Kip1 away from inhibition of cyclin E-Cdk2. The signaling mechanism by which DABK regulated p27Kip1 was explored. DABK was found to stimulate the activity of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) and to prolong activation of MEK and ERK by PDGF. Inhibition of ERK activation with the MEK inhibitors PD-98059 and U-0126 as well as the Src family kinase inhibitor PP2 completely blocked the effect of DABK to increase p27Kip1 and partially reversed the DABK-mediated inhibition of PDGF-stimulated proliferation. These studies demonstrate that the B1R inhibits PDGF-stimulated mitogenesis in part by prolonged activation of ERK leading to increased expression of p27Kip1.

The cardiovascular system is among the first organs formed during development and is pivotal for the formation and function of the rest of the organs and tissues. Therefore, the function and homeostasis of the cardiovascular system are finely regulated by many important molecules. Extensive studies have shown that platelet-derived growth factors (PDGFs) and their receptors are critical regulators of the cardiovascular system. Even though PDGF-C and PDGF-D are relatively new members of the PDGF family, their critical roles in the cardiovascular system as angiogenic and survival factors have been amply demonstrated. Understanding the functions of PDGF-C and PDGF-D and the signaling pathways involved may provide novel insights into both basic biomedical research and new therapeutic possibilities for the treatment of cardiovascular diseases.

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.

Liver fibrosis is a challenging global health problem resulting from chronic liver injury with no treatment currently available. It has been shown that activators for different peroxisome proliferator-activated receptor (PPAR) isoforms (α, γ, and δ) can affect different pathways in liver fibrosis. To evaluate the effects of the dual PPAR-α/γ agonist saroglitazar (SGZ) against thioacetamide (TAA)-induced fibrosis in rats, SGZ was administered for 6 weeks together with TAA injection. Administration of SGZ ameliorated TAA-induced elevation in hepatic biomarkers. SGZ was able to inhibit periportal and intralobular fibrous connective tissue proliferation, to decrease hydroxyproline content, and to lower alpha smooth muscle actin (α-SMA) protein expression. To unearth the antifibrotic mechanism of SGZ, the role of several fibrotic markers was studied. SGZ possesses inhibitory effect on protein levels of leptin, transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB). Furthermore, SGZ rectified matrix degradation through decreasing tissue inhibitor of metalloproteinases-1 (TIMP-1). This study suggests that SGZ could have a possible antifibrotic effect via suppression of leptin that can repress TGF-β1 and PDFG-BB, with subsequent inhibition of TIMP-1.

The role of PDGF-B and its receptor in meningeal tumorigenesis is not clear. We investigated the role of PDGF-B in mouse meningioma development by generating autocrine stimulation of the arachnoid through the platelet-derived growth factor receptor (PDGFR) using the RCAStv-a system. To specifically target arachnoid cells, the cells of origin of meningioma, we generated the PGDStv-a mouse (Prostaglandin D synthase). Forced expression of PDGF-B in arachnoid cells in vivo induced the formation of Grade I meningiomas in 27% of mice by 8 months of age. In vitro, PDGF-B overexpression in PGDS-positive arachnoid cells lead to increased proliferation.We found a correlation of PDGFR-B expression and NF2 inactivation in a cohort of human meningiomas, and we showed that, in mice, Nf2 loss and PDGF over-expression in arachnoid cells induced meningioma malignant transformation, with 40% of Grade II meningiomas. In these mice, additional loss of Cdkn2ab resulted in a higher incidence of malignant meningiomas with 60% of Grade II and 30% of Grade III meningiomas. These data suggest that chronic autocrine PDGF signaling can promote proliferation of arachnoid cells and is potentially sufficient to induce meningiomagenesis. Loss of Nf2 and Cdkn2ab have synergistic effects with PDGF-B overexpression promoting meningioma malignant transformation.

Healing of tendon ruptures represents a major challenge in musculoskeletal injuries and combinations of biomaterials with biological factors are suggested as viable option for improved healing. The standard approach of repair by conventional suture leads to incomplete healing or rerupture. Here, a new elastic type of DegraPol® (DP), a polyester urethane, is explored as a delivery device for platelet-derived growth factor-BB (PDGF-BB) to promote tendon healing. Using emulsion electrospinning as an easy method for incorporation of biomolecules within polymers, DegraPol® supports loading and release of PDGF-BB. Morphological, mechanical and delivery device properties of the bioactive DP scaffolds, as well as differences arising due to different electrospinning parameters are studied. Emulsion electrospun DP scaffolds result in thinner fibers than pure DP scaffolds and experience decreased strain at break [%], but high enough for successful surgeon handling. PDGF-BB is released in a sustained manner from emulsion electrospun DP, but not completely, with still large amount of it being inside the polymeric fibers after 30 d. In vitro studies show that the bioactive scaffolds promote tenocyte proliferation in serum free and serum(+) conditions, demonstrating the potential of this surgeon-friendly bioactive delivery device to be used for tendon repair.

Maple syrup urine disease (MSUD) is an inherited disorder caused by deficient activity of the branched-chain α-keto acid dehydrogenase complex involved in the degradation pathway of branched-chain amino acids (BCAAs) and their respective α-keto-acids. Patients affected by MSUD present severe neurological symptoms and brain abnormalities, whose pathophysiology is poorly known. However, preclinical studies have suggested alterations in markers involved with neurodegeneration. Because there are no studies in the literature that report the neurodegenerative markers in MSUD patients, the present study evaluated neurodegenerative markers (brain-derived neurotrophic factor (BDNF), cathepsin D, neural cell adhesion molecule (NCAM), plasminogen activator inhibitor-1 total (PAI-1 (total)), platelet-derived growth factor AA (PDGF-AA), PDGF-AB/BB) in plasma from 10 MSUD patients during dietary treatment. Our results showed a significant decrease in BDNF and PDGF-AA levels in MSUD patients. On the other hand, NCAM and cathepsin D levels were significantly greater in MSUD patients compared to the control group, while no significant changes were observed in the levels of PAI-1 (total) and PDGF-AB/BB between the control and MSUD groups. Our data show that MSUD patients present alterations in proteins involved in the neurodegenerative process. Thus, the present findings corroborate previous studies that demonstrated that neurotrophic factors and lysosomal proteases may contribute, along with other mechanisms, to the intellectual deficit and neurodegeneration observed in MSUD.

Physical exercise is recommended for a healthy lifestyle. Strenuous exercise, however, may trigger the haemostatic system, increasing the risk of vascular thrombotic events and the incidence of primary cardiac arrest. Our goal was to study the effects of strenuous exercise on risk factors of cardiovascular disease. Blood was collected from 92 healthy volunteers who participated in the amateur version of the pro-tour Amstel Gold cycling race, before and directly after the race. Thrombin generation showed a shortening of the lag time and time to peak and an increase of the velocity index. Interestingly, the endogenous thrombin potential measured in plasma decreased due to reduced prothrombin conversion. Platelet reactivity increased and this effect was stronger in men than in women. Lower fibrinogen and higher D-dimer levels after exercise indicated higher fibrin formation. On the other hand, fibrinolysis was also elevated as indicated by a shortening of the clot lysis time. Exercise activated the endothelium (von Willebrand factor (VWF) and active VWF levels were elevated) and the immune system (concentrations IL-6, IL-8, MCP-1, RANTES and PDGF increased). Additionally, an increased cardiac troponin T level was measured post-exercise. Strenuous exercise induces a temporary hyperreactive state in the body with enhanced pro- and anticoagulant responses. As strenuous exercise has a more pronounced effect on platelet function in male subjects, this gives a possible explanation for the higher incidence of sudden cardiac death during exercise compared to women. This trial is registered at www.clinicaltrials.gov as NCT02048462.

BACKGROUND

Revascularization of amputated extremities after prolonged ischemia is complicated by reperfusion injury. We assessed ischemia/reperfusion (I/R) injury of porcine extremities after prolonged preservation using extracorporeal circulation (ECC).

METHODS

Forelimbs of 32 pigs were divided into four groups based on ischemia times: group I: 6 h, group II: 12 h, group III: 0 h plus replantation, and group IV: 6 h plus replantation. Limbs were perfused with autologous blood using ECC for 12 h except group II with only 5 h perfusion. Limbs from groups III and IV were heterotopically replanted with a 7-d follow-up. Contralateral limbs served as controls in all groups. Tissue, plasma, and serum were analyzed for the extent of I/R injury.

RESULTS

No significant differences in tissue wet/dry ratios were found within or between groups. This finding was confirmed by histology, except for an increased damage in group IV muscles compared with baseline (P = 0.016). Complement C3 deposition was only increased in group IV muscle (P = 0.031), group II nerves (P = 0.046), and group II vessels (P = 0.037). Group IV muscle and nerve tissues were the only ones with significant IgM antibody deposition (P = 0.031) at end of perfusion. Values were normal again after replantation. Reduced complement activity and elevated IL-6, IL-8, MCP-1, VEGF, PDGF-bb, bFGF, and complement split products were found during perfusion but were normal again after replantation. Staining for heparin sulfate proteoglycans and von Willebrand factor confirmed minimal activation of endothelial cells.

CONCLUSIONS

The results demonstrate that prolonged limb preservation using ECC has minimal impact on I/R-induced tissue injury. Extracorporeal perfusion is a potential limb-preserving technique encouraging further studies for use in limb revascularization.

Protein kinase <em>D</em> (PK<em>D</em>) consists of a family of three structurally related enzymes that are co-expressed in the heart and have important roles in many biological responses. PK<em>D</em>1 is activated by pro-hypertrophic stimuli and has been implicated in adverse cardiac remodeling. Efforts to define the cardiac actions of PK<em>D</em>2 and PK<em>D</em>3 have been less successful at least in part because conventional methods provide a general screen for PK<em>D</em> activation but are poorly suited to resolve activation patterns for PK<em>D</em>2 or PK<em>D</em>3. This study uses Phos-tag S<em>D</em>S-PAGE, a method that exaggerates phosphorylation-dependent mobility shifts, to overcome this technical limitation. Phos-tag S<em>D</em>S-PAGE resolves PK<em>D</em>1 as distinct molecular species (indicative of pools of enzyme with distinct phosphorylation profiles) in unstimulated cardiac fibroblasts and cardiomyocytes; as a result, attempts to track PK<em>D</em>1 mobility shifts that result from agonist activation were only moderately successful. In contrast, PK<em>D</em>2 and PK<em>D</em>3 are recovered from resting cardiac fibroblasts and cardiomyocytes as single molecular species; both enzymes display robust mobility shifts in Phos-tag S<em>D</em>S-PAGE in response to treatment with sphingosine-1-phosphate, thrombin, <em>PDGF</em>, or H2O2. Studies with GF109203X implicate protein kinase C activity in the stimulus-dependent pathways that activate PK<em>D</em>2/PK<em>D</em>3 in both cardiac fibroblasts and cardiomyocytes. Studies with C3 toxin identify a novel role for Rho in the sphingosine-1-phosphate and thrombin receptor-dependent pathways that lead to the phosphorylation of PK<em>D</em>2/3 and the downstream substrate CREB in cardiomyocytes. In conclusion, Phos-tag S<em>D</em>S-PAGE provides a general screen for stimulus-specific changes in PK<em>D</em>2 and PK<em>D</em>3 phosphorylation and exposes a novel role for these enzymes in specific stress-dependent pathways that influence cardiac remodeling.

Platelet-derived growth factor (PDGF) represents one of the most prominent inducer of smooth muscle cell (SMC) migration and proliferation. Homo- and heterodimers of PDGF-A, PDGF-B, PDGF-C and PDGF-D subunits act by binding to homo- or heterodimers of the PDGF tyrosine kinase receptors, PDGFR-α and PDGFR-β. The essential role of PDGFR signaling on restenosis post-angioplasty or atherosclerosis has been demonstrated by using blocking antibodies to PDGF or the tyrosine kinase inhibitor imatinib. More specifically, molecular studies have defined the intracellular signaling pathways activated by PDGF, inducing the cell cycle progression and the migration of SMCs. Considering the relevant role of PDGF in atherogenesis, several studies have been performed to investigate the effect of naturally occurring compounds on both in vitro and in vivo experimental models of atherogenesis. The present review will briefly summarize the pathophysiological role of PDGF and the studies of natural inhibitors tested in in vivo experimental models of restenosis in response to vascular injury and/or atherosclerosis.

BACKGROUND

Platelet-derived growth factor D (PDGF-D) plays an important role in angiogenesis, vessel remodeling, inflammation and repair in response to injury. We hypothesized that genetic variation in PDGFD gene might alter the susceptibility to stroke.

RESULTS

We determined the genotypes of a single nucleotide polymorphism (SNP) (-858A/C, rs3809021) in 1484 patients with stroke (654 cerebral thrombosis, 419 lacunar infarction, 411 intracerebral hemorrhage [ICH]) and 1528 control subjects from an unrelated Chinese Han population and followed the stroke patients up for a median of 4.5 years.The -858AA genotype showed significantly increased risk of ICH (dominant model: odds ratio [OR] 1.29, 95% confidence interval [CI] 1.00-1.68, P = 0.05; additive model: OR 1.24, 95% CI 1.01-1.52, P = 0.04) than wild-type genotype. Further analyses showed that -858AA genotype conferred about 2-fold increase in risk of non-hypertensive ICH (dominant model: OR 2.1, 95%CI 1.34-3.29, P = 0.001; additive model: OR 1.75, 95% CI 1.24-2.46, P = 0.001). After a median follow-up of 4.5 years, -858AA genotype was associated with a reduced risk of ICH recurrence (dominant model: adjusted hazard ratio [HR] 0.09, 95%CI 0.01-0.74, P = 0.025; additive model: HR 0.21, 95% CI 0.04-1.16, P = 0.073) in non-hypertensive patients.

CONCLUSIONS

The -858AA genotype is probably associated with risk for non-hypertensive ICH. Further studies should be conducted to reveal the role of PDGF-D at various stages of ICH development--beneficial, or deleterious.

BACKGROUND

Neointimal hyperplasia involves the migration of medial vascular smooth muscle cells (VSMCs) in response to arterial injury. Thrombospondin-1 (TSP1), platelet-derived growth factor (PDGF), and fibronectin (Fn) induce VSMC migration. Nitric oxide (NO) limits VSMC migration. The hypothesis of this study is that NO would dose dependently inhibit TSP1-induced, PDGF-induced, and Fn-induced VSMC chemotaxis.

METHODS

VSMCs were pretreated with serum free media or the NO donors diethylenetriamine NONOate or S-nitroso-N-acetyl-D,L-penicillamine. Chemotaxis to TSP1, PDGF, or Fn was determined. Analysis of variance with post hoc testing was done. P values < .05 were considered significant.

RESULTS

PDGF, TSP1, and Fn induced VSMC chemotaxis. NO donors inhibited chemotaxis of VSMCs to PDGF in a concentration-dependent manner. NO donors had a variable effect on TSP1-induced chemotaxis. NO donors did not inhibit Fn-induced chemotaxis.

CONCLUSIONS

The complex interactions of these proteins in vivo will need to be considered when developing NO-dependent therapies for neointimal hyperplasia.

LIGHT (HVEM-L, TNFSF14, or CDD for herpes virus entry mediator (HVEM)/tumor necrosis factor (TNF)-related 2, is a member of the TNF superfamily. It is expressed as a homotrimer on activated T cells and dendritic cells (DCs), and has three receptors: HVEM, LT-β receptor (LTβR), and decoy receptor 3 (DcR3). So far, three receptors with distinct cellular expression patterns are known to interact with LIGHT. Follicular DCs and stromal cells bind LIGHT through LTβR. We monitored the effects of LIGHT on human bone marrow-derived mesenchymal stem cells (BM-MSCs). At first, we checked the negative and positive differentiation markers of BM-MSCs. And we confirmed the quality of MSCs by staining cells undergoing adipogenesis (Oil Red O staining), chondrogenesis (Alcian blue staining), and osteogenesis (Alizarin red staining). After rhLIGHT treatment, we monitored the count, viability, and proliferation of cells and cell cycle distribution. PDGF and TGFβ production by rhLIGHT was examined by ELISA, and the underlying biological mechanisms were studied by immunoblotting by rhLIGHT treatment. LTβR was constitutively expressed on the surface of human BM-MSCs. Cell number and viability increased after rhLIGHT treatment. BM-MSC proliferation was induced by an increase in the S/G2/M phase. The expression of not only diverse cyclins such as cyclin B1, DDDK1 and CDK2, increased, while that of p27 decreased, after rhLIGHT treatment. RhLIGHT-induced PDGF and TGFβ production mediated by STAT3 and Smad3 activation accelerated BM-MSC proliferation. Thus, LIGHT and LTβR interaction increases the survival and proliferation of human BM-MSCs, and therefore, LIGHT might play an important role in stem cell therapy.

Background: The pathogenesis of central serous chorioretinopathy (CSC) remains a subject of intensive research. We aimed to determine correlations between plasma levels of selected angiogenic factors and different forms of CSC.

Methods: Eighty patients were enrolled in the study including 30 with a chronic form of CSC, 30 with acute CSC, and 20 controls. Presence of active CSC was determined by fluorescein angiography (FA), indocyanine green angiography (ICGA), and swept-source optical coherence tomography (SS-OCT). Plasma concentrations of angiopoietin-1, endostatin, fibroblast growth factor, placental growth factor (PlGF), platelet-derived growth factor (PDGF-AA), thrombospondin-2, vascular endothelial growth factor (VEGF), VEGF-D, and pigment epithelium-derived factor were measured, and the results were compared between groups. Additionally, mean choroidal thickness (CT) was measured in all patients.

Results: Levels of angiopoietin-1 (p = 0.008), PlGF (p = 0.045), and PDGF-AA (p = 0.033) differed significantly between the three groups. Compared with the controls, VEGF (p = 0.024), PlGF (p = 0.013), and PDGF-AA (p = 0.012) were downregulated in the whole CSC group, specifically PDGF-AA (p = 0.002) in acute CSC and angiopoietin-1 (p = 0.007) in chronic CSC. An inverse correlation between mean CT and VEGF levels was noted in CSC patients (rho = -0.27, p = 0.044).

Conclusions: Downregulated angiopoietin-1, VEGF, PDGF-AA, and PlGF levels may highlight the previously unknown role of the imbalanced levels of proangiogenic and antiangiogenic factors in the pathogenesis of CSC. Moreover, downregulated VEGF levels may suggest that choroidal neovascularization in CSC is associated with arteriogenesis rather than angiogenesis.

Keywords: angiogenesis; angiopoietin-1; central serous chorioretinopathy; proangiogenic factors.

OBJECTIVE

Wound healing is an orderly complex process involving inflammation, clotting, re-epithelialization, neovascularization and wound closure. In diabetic patients, such process is impaired and delayed, posing negative economic as well as social consequences. Diabetex, (patency# EP 0877617 A1) composed of L-alanine, d-ribose, nicotinic acid and calcium ascorbate, which was initially introduced to treat cancer is thought to have anti- diabetic effects. The present study was designed to investigate the therapeutic merit of diabetex as well as the cellular mechanisms involved in such effects and its safety profile compared to metformin in wounded diabetic rats.

METHODS

Sixty adult male Sprague-Dawley albino rats were randomly divided into two major groups after induction of full thickness wound; control and treated groups. Liver and kidney function test, as well as cytokines (VEGF, TGF-β, PDGF and MMP2), fasting blood sugar were measured in animal sera. Histopathological studies including hematoxyline and eosin, Masson's trichrome stains were performed on wounded tissue.

RESULTS

Diabetex significantly improved wound healing, collagen formation, induced re-epithelialization and neovascularization. Moreover, cytokines involved in wound healing process were increased by the antidiabetic medication. Noteworthy, the drug exhibited a safe profile on liver and kidney function tests and significantly reduced fasting blood sugar.

CONCLUSIONS

The present study offers a novel approach for treating diabetic resistant wounds with a possible more economic, safe strategy.

Arsenic trioxide (As2O3) exhibits a remarkable effect on leukemia treatment; however, its effect on solid tumors remains poorly explored. The present study demonstrated the inhibitory effect of As2O3 on lung cancer and explored its possible mechanism. It was observed that As2O3 significantly inhibited the growth of lung cancer xenografts and tumor angiogenesis in vivo. The inhibitory effect of As2O3 on cell proliferation in vitro was more remarkable in vascular endothelial cells than in lung cancer cells. It was also observed that As2O3 inhibited the migration of vascular endothelial cells and disrupted vascular tube formation on Matrigel assays. In addition, a series of key signaling factors involved in multiple stages of angiogenesis, including matrix metalloproteinase (MMP)-2, MMP-9, platelet-derived growth factor (PDGF)-BB/PDGF receptor-β, vascular endothelial growth factor (VEGF)-A/VEGF receptor-2, basic fibroblast growth factor (FGF)/FGF receptor-1 and delta like canonical Notch ligand 4/Notch-1, were regulated by As2O3. These findings suggested that anti-angiogenesis may be an underlying mechanism of As2O3 anticancer activity in lung cancer.

There is emerging interest in the use of standardized virally inactivated human platelet lysate preparations rich in GFs (growth factors) for cell cultures, cell therapy and clinical applications. In the present paper, we report a simple process to prepare a virally inactivated platelet lysate preparation rich in TGF-beta1 (transforming growth factor-beta1), EGF (epidermal growth factor) and IGF (insulin-like growth factor) and depleted of PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor). Apheresis platelet concentrates were treated by the S/D (solvent/detergent) viral inactivation procedure, then subjected to an oil extraction followed by adsorption with activated charcoal and finally sterile-filtered. The resulting preparation contained a mean of 368.4, 2.4 and 54.7 ng/ml of TGF-beta1, EGF and IGF respectively. PDGF-AB and VEGF were essentially completely removed by the charcoal treatment. The mean albumin, IgG, IgM and IgA and fibrinogen contents were approx. 40.0, 8.5, 0.87, 1.66 and 2.65 mg/ml respectively, cholesterol and triglycerides were at 15 and 20.7 mg/ml respectively and TnBP (tri-n-butyl phosphate) and Triton X-45 were at 8.7 and 8.8 p.p.m. respectively. Supplementing MEM (minimum essential medium) with 1-10% of this S/D-treated platelet lysate promoted the proliferation of MG63 and SIRC cell lines as well as, or better than, 10% (v/v) FBS (fetal bovine serum), as based on the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The process used to prepare such S/D-treated platelet lysates is easily scalable for industrial production. Our results open up the possibility to evaluate the potential of this new preparation for stem cell expansion and/or bone tissue engineering and regeneration.

The temporal trajectory of platelet-derived growth factor (PDGF)-beta receptor activation within the dorsocaudal brainstem parallels that of the mild hypoxic ventilatory depression (HVD) seen in adult rats. We hypothesized that enhanced PDGF-beta receptor activity may account for the particularly prominent HVD of developing mammals. To study this issue, 2-, 5-, 10-, and 20-d-old rats underwent hypoxic challenges (10% O(2) for 30 min) after pretreatment with either vehicle (Veh) or the selective PDGF-beta receptor antagonist CGP57148B (intraperitoneal 100 mg/kg). The developmental characteristics and magnitude of the peak hypoxic ventilatory response (HVR) were not modified by the PDGF-beta receptor blocker. However, HVD was markedly attenuated by CGP57148B, and such effect, although still present, gradually abated with increasing postnatal age [p < 0.001, analysis of variance (ANOVA)]. Hypercapnic ventilatory responses were not affected by CGP57148B. The expression of PDGF-beta receptor in the dorsocaudal brainstem was assessed by immunoblotting and confirmed progressively decreasing expression with maturation. We conclude that PDGF-beta receptor activation during hypoxia is an important contributor to HVD at all postnatal ages but more particularly in the immature rat.

Smooth muscle cells (SMCs) play critical roles in a number of diseases; however, the molecular mechanism underlying their development is unclear. Although the role of TGFβ1 signaling in SMC development is well established, the downstream molecular signals are not fully understood. We used several rat multipotent adult progenitor cell ((r)MAPC) lines that express levels of Oct4 mRNA similar to hypoblast stem cells (HypoSC), and can differentiate robustly to mesodermal and endodermal cell types. TGFβ1 alone, or with <em>PDGF</em>-BB, induces differentiation of rMAPCs to SMCs, which expressed structural SMC proteins, including α-smooth muscle actin (αSMA), and contribute to the SMC coat of blood vessels in vivo. A genome-wide time-course transcriptome analysis revealed that transcripts of Baf60c, part of the SWI/SNF actin binding chromatin remodeling complex <em>D</em>-3 (SMARC<em>D</em>3/BAF60c), were significantly induced during MAPC-SMC differentiation. We demonstrated that BAF60c is a necessary co-regulator of TGFβ1 mediated induction of SMC genes. Knock-down of Baf60c decreased SMC gene expression in rMAPCs whereas ectopic expression of Baf60c was sufficient to commit rMAPCs to SMCs in the absence of exogenous cytokines. TGFβ1 activates Baf60c via the direct binding of SMA<em>D</em>2/3 complexes to the Baf60c promoter region. Chromatin- and co-immunoprecipitation studies demonstrated that regulation of SMC genes by BAF60c is mediated via interaction with SRF binding CArG box-containing promoter elements in SMC genes. We noted that compared with TGFβ1, Baf60c overexpression in rMAPC yielded SMC with a more immature phenotype. Similarly, Baf60c induced an immature phenotype in rat aortic SMCs marked by increased cell proliferation and decreased contractile marker expression. Thus, Baf60c is important for TGFβ-mediated commitment of primitive stem cells (rMAPCs) to SMCs and is associated with induction of a proliferative state of quiescent SMCs. The MAPC-SMC differentiation system may be useful for identification of additional critical (co-)regulators of SMC development.

Three specific platelet-derived growth factor (PDGF) isoforms are thought to bind with differing affinities to two distinct PDGF receptors which undergo activation following dimerization. Recent evidence has been presented that marked differences exist between the ability of PDGF-AA versus PDGF-AB and PDGF-BB to stimulate alterations in second messengers in cultures of vascular smooth muscle cells (VSMC), a result which was thought to be due to low numbers of the A-type receptor in this cell type (Sachinidis, A., Locker, R., Vetter, W., Tatje, D., and Hoppe, J. (1990) J. Biol. Chem. 265, 10238-10243, 1990). In particular, PDGF-BB and PDGF-AB but not PDGF-AA could elicit alterations in cytosolic free calcium (Ca2+i). However, because these studies were performed on large cell populations using biochemical assays of PDGF activity, a minor PDGF-AA-Ca(2+)-responsive population of cells might go undetected. To test this possibility, VSMC were isolated from either thoracic or abdominal pig aorta, and alterations in Ca2+i were monitored using Multiparameter Digitized Video Microscopy following stimulation with PDGF isoforms alone, or either before or after exposure of VSMC to 5 mM EGTA. PDGF-AA-responsive cells were found to exist only in cultures of thoracic VSMC, caused oscillations in Ca2+i, represented 20% of the PDGF-BB-responsive cells, and were subsequently responsive to PDGF-BB. PDGF-BB elicited monophasic alterations in Ca2+i in both thoracic and abdominal VSMC. Prior addition of EGTA inhibited PDGF-AA but not PDGF-BB-induced alterations in Ca2+i. Addition of EGTA during PDGF-AA-induced Ca2+i oscillations inhibited subsequent oscillations in Ca2+i, while addition of EGTA at the peak of the PDGF-BB Ca2+ response resulted in a more rapid return of Ca2+i to prestimulation levels. These data suggest that regional differences in the distribution of PDGF-A- and B-type receptor exists in vivo, and that activation of the A- and B-type PDGF receptors results in distinct alterations in Ca2+i.

Platelet-derived growth factor (PDGF) BB is a mitogen that stimulates bone resorption and increases collagenase 3 transcription in osteoblasts, although the mechanisms involved are as yet unknown. We examined the effect of PDGF BB on collagenase 3 transcription in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased the activity of collagenase 3 promoter fragments transiently transfected into Ob cells. Deletion analysis of the collagenase promoter revealed three regions that impaired the induction of collagenase 3 by PDGF BB. A construct spanning base pair -53 to +28 collagenase 3 sequences, in relation to the start site of transcription +1, was fully responsive to PDGF BB and was studied in detail. Targeted mutations of an AP-1 site in this fragment decreased basal collagenase promoter activity and the responsiveness to PDGF BB, whereas mutations of Stat3 and Ets binding sites did not alter the response to PDGF. Electrophoretic mobility shift assay, using nuclear extracts from control and treated cells, revealed AP-1 nuclear protein complexes that were enhanced in extracts from PDGF BB-treated Ob cells. Supershift assays revealed that antibodies to c-Fos, Fos B, Fra-2, c-Jun, Jun B, and Jun D shifted the binding of nuclear extracts from cells treated with PDGF BB to AP-1 sequences. In conclusion, PDGF BB induces collagenase 3 transcription in osteoblasts by regulating nuclear proteins interacting with AP-1 sequences.