OBJECTIVE

Polycystic ovary syndrome (PCOS), characterized by female infertility and metabolic abnormalities, is one of the most common endocrine disorders. The etiology of PCOS remains unknown. The comprehensive analysis of protein alterations in PCOS patients is meaningful for identifying diagnostic biomarkers of PCOS. Here, we explored the clinical value of serum proteins as novel biomarkers to detect PCOS with low progesterone level.

METHODS

A total of 43 patients with PCOS and 30 healthy women were enrolled. Protein array was used to detect the variations of serum proteins between PCOS patients and healthy women. The level of five serum proteins was further confirmed by ELISA and western blot. The human ovarian granulosa cells (KGN) was cultured to examine the underlying mechanism of PCOS. CCK8 assay and western blot were carried out to evaluate the alterations in proliferative ability, TUNEL assay and DAPI staining to detect the apoptosis of KGN cells.

RESULTS

Among the 507 proteins, we identified 76 differentially expressed serum proteins (≧1.5 fold), with 40 elevated and 36 decreased proteins. Moreover, 47 proteins were newly reported in PCOS. The alterations in the five significantly decreased proteins (EREG, inhibin βA, IDE, PDGF-D and KNG1) were further confirmed by ELISA and western blot. The level of these proteins were directly associated with the low progesterone, and the expression could be upregulated by progesterone. EREG and inhibin βA also promoted the proliferation and inhibited the apoptosis of ovarian granulosa cells.

CONCLUSIONS

The study highlights that serum proteins are differentially expressed in PCOS patients and healthy women, and EREG and inhibin βA levels are upregulated by progesterone, which are correlated with ovarian functions. The study suggests that EREG and inhibin βA may be applied as novel potential biomarkers for PCOS with low progesterone level.

The peptides platelet-derived growth factor-A (PDGF-A) and especially -B have important roles in lung development. The effect of hyperoxic exposure with and without inhaled nitric oxide (iNO) on lung expression of PDGF and its receptors is unknown. We hypothesized that hyperoxia exposure would suppress mRNA expression and protein production of these ligands and their receptors. The addition of iNO to hyperoxia may further aggravate the effects of hyperoxia. Thirteen-day-old piglets were randomized to breathe 1) room air (RA); 2) 0.96 fraction of inspired oxygen (O2), or 3) 0.96 fraction of inspired oxygen plus 50 ppm of NO (O2+NO), for 5 d. Lungs were preserved for mRNA, Western immunoblot, and immunohistochemical analyses for PDGF-A and -B and their receptors PDGFR-alpha and -beta. PDGF-B mRNA expression was greater than that of PDGF-A or PDGFR-alpha and -beta in RA piglet lungs (p<0.05). Hyperoxia with or without iNO reduced lung PDGF-B mRNA and protein expression relative to the RA group lungs (p<0.01). PDGF-B immunostain intensity was significantly increased in the alveolar macrophages, which were present in greater numbers in the hyperoxia-exposed piglet lungs, with or without NO (p<0.01). PDGFR-beta immunostaining was significantly increased in airway epithelial cells in O2- and O2+NO-exposed piglets. PDGF-A and PDGFR-alpha immunostain intensity and distribution pattern were unchanged relative to the RA group. Sublethal hyperoxia decreases PDGF-B mRNA and protein expression but not PDGF-A or their receptors in piglet lungs. iNO neither aggravates nor ameliorates this effect.

Extensive proliferation of vascular smooth muscle cell (VSMC) contributes to intimal hyperplasia following vascular injury, in which endoplasmic reticulum stress (ERS) plays a critical role. Intermedin (IMD) is a vascular paracrine/autocrine peptide exerting numerous beneficial effects in cardiovascular diseases. IMD overexpression could alleviate intimal hyperplasia. Here, we investigated whether endogenous IMD protects against intimal hyperplasia by inhibiting endoplasmic reticulum stress. The mouse left common carotid-artery ligation-injury model was established to induce intimal hyperplasia using IMD^-/-mice and C57BL/6 J wild-type (WT) mice. Platelet-derived growth factor-BB (PDGF-BB) was used to stimulate the proliferation of VSMC. IMD^-/- mice displayed exacerbated intimal hyperplasia induced by complete ligation of the left carotid artery at 14 d and 28 d compared to WT mice. However, IMD-deficiency had no effect on blood pressure, plasma triglyceride, and fasting blood glucose levels in mice. Furthermore, VSMCs derived from IMD^-/- mice showed increased cell proliferation and dramatically elevated levels of glucose regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), ATF6 mRNA under PDGF-BB treatment compared to WT mice-derived VSMCs. In addition, exogenous administration of IMD significantly attenuated PDGF-BB-induced cell proliferation and GRP78, phosphorylase-inositol requiring enzyme 1α, ATF4, and ATF6 protein levels. Thus, endogenous IMD may counteract ERS to exert protective role in response to vascular injury and IMD is expected to be a therapeutic target for the prevention and treatment of restenosis.

The success of periodontal regeneration depends on the coordination of early cell proliferation and late cell differentiation. The aim of this study was to investigate whether the proliferation or differentiation stage predominantly promotes the initiation of periodontal regeneration. Critical-sized periodontal defects were surgically created on rat maxillae and filled with poly-(D,L-lactide-co-glycolide)-poly-d,l-lactide hybrid microspheres encapsulating platelet-derived growth factor (PDGF, a promoter of mitogenesis), simvastatin (a promoter of osteogenic differentiation), or bovine serum albumin (a control). The encapsulation efficiency and in vitro release profiles of the microspheres were determined by high-performance liquid chromatography and enzyme-linked immunosorbent assay. The maxillae were harvested after 10 or 14 days and assessed by micro-computed tomography, histology, and immunohistochemistry for regeneration efficacy and cell viability. The rapid release of PDGF was observed within the first week, whereas a slow release profile was noted for simvastatin. The PDGF-treated specimens demonstrated a significantly higher bone volume fraction compared with bovine serum albumin- (p < 0.05) or simvastatin-treated (p < 0.05) specimens at day 14. Histologically, active bone formation originating from the defect borders was noted in both the PDGF- and the simvastatin-treated specimens, and functionally aligned periodontal ligament fiber insertion was only observed in the PDGF-treated specimens. The significant promotion of mitogenesis by PDGF treatment was also noted at day 14 (p < 0.05). In conclusion, increased mitogenesis or osteogenic differentiation may stimulate osteogenesis, and the upregulation of mitogenesis by PDGF appears to play a role in the initiation of periodontal regeneration.

OBJECTIVE

Mouse mammary cancer cell line MCH66 shows invasion-independent metastasis. To elucidate this metastatic mechanism, the biological characteristics putatively related to metastasis were analyzed using several cell lines with different metastatic abilities derived from MCH66.

METHODS

Metastatic capacity, invasive activity, growth property, and mRNA expressions of factors associated with endothelial cell proliferation were comparatively analyzed in MCH66 and its sublines.

RESULTS

Lu10 subline exhibited higher metastatic potential to the lungs and lymph nodes (100%) than MCH66 or Lu1 subline (0/5, 0/5 each). The growth rate was almost identical between Lu10 and MCH66, and Lu10 revealed weaker invasive activity in vitro than MCH66. In Lu10 tumors in mice, well-developed sinusoidal blood vessels and dilated lymphatics were noted compared with in Lu1 tumors. Accordingly, Lu10 showed higher expression of vascular endothelial growth factor (VEGF)-C, -D, platelet-derived growth factor (PDGF)-B and pleiotrophin than Lu1, while the expression of other growth factors such as VEGF-A, midkine, angiogenin, hepatocyte growth factor, PDGF-A, and basic fibroblast growth factor remained unchanged between Lu1 and Lu10.

CONCLUSIONS

These data indicate that high invasiveness and rapid growth are not required for this metastatic process, and some angiogenic mediators are involved in blood-borne and lymphatic metastasis.

Glutaric acidemia type I (GA1) is caused by severe deficiency of glutaryl-CoA dehydrogenase activity, resulting in an accumulation of glutaric acid and glutarylcarnitine (C5DC) in the organism. Patients affected by GA1 are asymptomatic in the neonate period but usually manifest chronically progressive neurodegeneration apart from severe encephalopathic crises associated with acute striatum necrosis. Neurological manifestations like dyskinesia, dystonia, hypotonia, muscle stiffness and spasticity are present. Treatment is based on protein/lysine restriction and L-carnitine supplementation. In this work we evaluated markers of neurodegeneration and inflammation, namely BDNF (brain-derived neurotrophic factor), NCAM (neuronal adhesion molecule), PDGF-AA (platelet-derived growth factor) and cathepsin-D in plasma of six treated GA1 patients. We first found marked increases of plasma C5DC concentrations in GA1 patients, as well as increased levels of the markers BDNF and cathepsin-D as compared to those of age-matched healthy children. Furthermore, C5DC concentrations were highly correlated with the levels of cathepsin-D. These results may demonstrate that brain tissue degeneration is present in GA1 patients and that there is a relationship between increased metabolites concentrations with this process. To the best of our knowledge this is so far the first study showing altered peripheral parameters of neurodegeneration and inflammation in GA1 patients.

The oncogenic roles of PDGF-D and its proteolytic activator, matriptase, have been strongly implicated in human prostate cancer. Latent full-length PDGF-D (FL-D) consists of a CUB domain, a growth factor domain (GFD), and the hinge region in between. Matriptase processes the FL-D dimer into a GFD dimer (GFD-D) in a stepwise manner, involving generation of a hemidimer (HD), an intermediate product containing one FL-D subunit and one GFD subunit. Although the HD is a pro-growth factor that can be processed into the GFD-D by matriptase, the HD can also act as a dominant-negative ligand that prevents PDGF-B-mediated β-PDGF receptor activation in fibroblasts. The active GFD-D can be further cleaved into a smaller and yet inactive form if matriptase-mediated proteolysis persists. Through mutagenesis and functional analyses, we found that the R(340)R(341)GR(343)A (P4-P1/P1') motif within the GFD is the matriptase cleavage site through which matriptase can deactivate PDGF-D. Comparative sequence analysis based on the published crystal structure of PDGF-B predicted that the matriptase cleavage site R(340)R(341)GR(343)A is within loop III of the GFD, a critical structural element for its binding with the β-PDGF receptor. Interestingly, we also found that matriptase processing regulates the deposition of PDGF-D dimer species into the extracellular matrix (ECM) with increased binding from the FL-D dimer, to the HD, and to the GFD-D. Furthermore, we provide evidence that R(340)R(341)GR(343)A within the GFD is critical for PDGF-D deposition and binding to the ECM. In this study, we report a structural element crucial for the biological function and ECM deposition of PDGF-D and provide molecular insight into the dynamic functional interplay between the serine protease matriptase and PDGF-D.

Contexts:Sauromatum guttatum (Wall.) Schott (Araceae) has been traditionally used for the treatment of wounds. Objectives: This study evaluates the healing and tissue regeneration potential of S. guttatum extract in burn wounds. Materials and methods:S. guttatum extract was analysed using various chemical tests, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Moreover, the extract was tested against burn associated bacteria and minimum inhibitory concentration (MIC) was also calculated. Wound healing and tissue regeneration potential was assessed using a thermally induced burn BALBc mouse model. S. guttatum extract (2% w/w) prepared in petroleum jelly, vehicle and positive control [silver sulfadiazine (SD)] groups was applied three times a day. The treatment was continued for 15 d and wound closure was measured and photographed on day 5, 10 and 15. The burnt tissues excised from wounds were subjected to histological and comparative gene expression analysis. Results: The results of the chemical tests indicated the presence of alkaloids, saponins, phenols, phytosterols, tannins, and flavonoids, while TLC and HPLC analysis indicated the presence of various compounds. The extract showed excellent activity against the tested pathogens. The lowest MIC (125 µg/mL) was observed against Staphylococcus aureus. A considerable decrease in wound area (72%) was observed in extract-treated group. Histological examination of extract-treated group showed good signs of wound healing with complete re-epithelialization and better tissue regeneration. Comparative gene expression analysis revealed the up-regulation of wound healing related PDGF, EGF and FGF genes. Conclusions:S. guttatum extract may be used to isolate bioactive constituents for the treatment of burn wounds.

Context: Terrestrosin D (TED), from Tribulus terrestris L. (Zygophyllaceae), exhibits anti-tumour and anti-inflammatory activities. However, its effects on bleomycin (BLM)-induced pulmonary inflammation and the subsequent fibrotic changes remain unclear. Objective: To examine the anti-inflammatory and anti-fibrotic effects of TED against BLM in murine pulmonary tissues. Materials and methods: Male SPF mice received saline (control), TED (10 mg/kg), BLM (2.5 mg/kg), or BLM (2.5 mg/kg) + TED (10 mg/kg) group. BLM was administered as a single intranasal inoculation, and TED was intraperitoneally administered once daily. After 2 and 6 weeks of treatment, cell number and differentiation (Giemsa staining) and TNF-α, IL-6, IL-8, TGF-β1, and PDGF-AB levels (ELISA) were determined in the bronchoalveolar lavage fluid (BALF). Hydroxyproline (Hyp) content in the left pulmonary tissue was also determined (ELISA). The right pulmonary tissue was H&E-stained and assessed for the severity of pulmonary fibrosis using the Ashcroft scoring method. Compared with the BLM group, TED decreased inflammatory cell infiltration; number of macrophages (p < 0.05), neutrophils (p < 0.05), lymphocytes (p < 0.05); percentage of macrophages in the monocyte-macrophage system (p < 0.05), and levels of TNF-α (p < 0.01), IL-6 (p < 0.01), IL-8 (p < 0.05), TGF-β1 (p < 0.05), and PDGF-AB (p < 0.05) in the BALF. TED also reduced Hyp content (p < 0.05) in the pulmonary tissue and attenuated the BLM-induced deterioration in lung histopathology. Discussion and conclusions: TED can inhibit BLM-induced inflammation and fibrosis in the lungs of mice, which may be related to reduced inflammatory and fibrotic markers. These results could be further tested in humans through clinical studies.

Targeted delivery of therapeutic agents to prevent smooth muscle cell (SMC) proliferation is important in averting restenosis (a narrowing of blood vessels). Since platelet derived growth factor (PDGF) receptors are over-expressed in proliferating SMCs after injury from cardiovascular interventions, such as angioplasty and stent implantation, our hypothesis is that conjugation of PDGF-BB (platelet-derived growth factor BB (homodimer)) peptides to biodegradable poly (D,L-lactic-co-glycolide) (PLGA) nanoparticles (NPs) would exhibit an increased uptake of these NPs by proliferating SMCs. In this study, poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles containing dexamethasone were formulated and conjugated with PDGF-BB peptides. These NPs were stable, biocompatible, and exhibited a sustained drug release over 14 days. Various particle uptake studies using HASMCs (human aortic smooth muscle cells) demonstrated that PDGF-BB peptide-conjugated nanoparticles significantly increased cellular uptake and decreased proliferation of HASMCs compared to control nanoparticles (without conjugation of PDGF-BB peptides). These NPs were internalized primarily by clathrin-mediated endocytosis and macropinocytosis. Our in vitro results suggest that PDGF-BB peptide-conjugated NPs could represent as an effective targeted, sustained therapeutic delivery system to reduce restenosis and neointimal hyperplasia.

This study reviewed some new aspects of the modular proteoglycan perlecan, a colossal proteoglycan with a 467 kDa core protein and five distinct functional domains. Perlecan is a heparan sulphate proteoglycan that transiently displays native CS sulphation motifs 4-C-3 and 7-D-4 during tissue morphogenesis these are expressed by progenitor cell populations during tissue development. Perlecan is susceptible to fragmentation by proteases during tissue development and in pathological tissues particularly in domains IV and V. The fragmentation pattern of domain IV has been suggested as a means of grading prostate cancer. Domain V of perlecan is of interest due to its interactive properties with integrin α5β1 that promotes pericyte migration enhancing PDGF-BB-induced phosphorylation of PDGFRβ, Src homology region 2 domain-containing phosphatase-2, and focal adhesion kinase supporting the repair of the blood brain barrier following ischaemic stroke. Fragments of domain V can also interact with α2β1 integrin disrupting tube formation by endothelial cells. LG1-LG2, LG3 fragments can antagonise VEGFR2, and α2β1 integrin interactions preventing angiogenesis by endothelial cells. These domain V fragments are of interest as potential anti-tumour agents. Perlecan attached to the luminal surfaces of endothelial cells in blood vessels acts as a flow sensor that signals back to endothelial and smooth muscle cells to regulate vascular tone and blood pressure. Perlecan also acts as a flow sensor in the lacuno-canalicular space regulating osteocytes and bone homeostasis. Along with its biomechanical regulatory properties in cartilaginous tissues this further extends the functional repertoire of this amazingly diverse functional proteoglycan.

Keywords: Perlecan; Tissue morphogenesis; cartilage; heparan sulphate; intervertebral disc; matricryptin; repair biology.

Platelet-derived growth factor receptor alpha (PDGFRalpha) interacts with PDGFs A, B, C and AB, while PDGFRbeta binds to PDGFs B and D, thus suggesting that PDGFRalpha is more promiscuous than PDGFRbeta. The structural analysis of PDGFRalpha-PDGFA and PDGFRalpha-PDGFB complexes and a molecular explanation for the promiscuity of PDGFRalpha and the specificity of PDGFRbeta remain unclear. In the present study, we modeled the three extracellular domains of PDGFRalpha using a previous crystallographic structure of PDGFRbeta as a template. Additionally, we analyzed the interacting residues of PDGFRalpha-PDGFA and PDGFRalpha-PDGFB complexes using docking simulations. The validation of the resulting complexes was evaluated by molecular dynamics simulations. Structural analysis revealed that changes of non-aromatic amino acids in PDGFRalpha to aromatic amino acids in PDGFRbeta (I139F, P267F and N204Y) may be involved in the promiscuity of PDGFRalpha. Indeed, substitution of amino acids with few probabilities of rotamer changes in PDGFRbeta (M133A, N163E and N179S) and energy stability due to the formation of hydrogen bond in PDGFRbeta could explain the specificity of PDGFRbeta. These results may be used as an input for a better and more specific drug and peptide design targeting diseases related with the malfunction of PDGFs and PDGFRalpha such as cancer and atherosclerosis.

Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with ovarian stimulation. OHSS features are ovarian enlargement with fluid shifting to the third space. Disturbances in the vasculature are considered the main changes that lead to OHSS. Our aim was to analyze the levels of angiopoietins 1 and 2 (ANGPT1 and 2) and their soluble and membrane receptors (s/mTie-2) in follicular fluid (FF) and in granulosa-lutein cells culture (GLCs) from women at risk of developing OHSS. We also evaluated the effect of ANGPT1 on endothelial cell migration. In ovaries from an OHSS rat model, we analyzed the protein concentration of ANGPTs, their mTie-2 receptor, and platelet-derived growth factor PDGF-B, -D and PDGFR-β. ANGPT1 levels were increased in both FF and GLCs from women at risk of OHSS. Incubation of these FF with an ANGPT1 neutralizing antibody decreased endothelial cell migration. In the ovaries of OHSS rat model, mTie-2 protein levels increased and PDGF-B and -D decreased. In summary, these results suggest that ANGPT1 could be another mediator in the development of OHSS.

Aims: Stem cell therapy has shown promise for treating myocardial infarction (MI) via re-muscularization and paracrine signaling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischemic conditions in the infarcted heart under these two different delivery approaches remain unclear. Here we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level.

Methods and results: NHP iPSC-CMs in both monolayer culture (2 D) and engineered heart tissue (EHT) (3 D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3 D EHT model was more sensitive to ischemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signaling.

Conclusions: By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischemic injury.

Translational perspective: Stem cell therapy has shown promise for treating ischemic heart tissue. However, how stem cells respond following different delivery method is unclear. Here hypoxic conditioning was applied to non-human primate iPSC-CMs in 2 D monolayer culture and 3 D engineered heart tissue to model cell injection versus patch implantation, respectively, in an ischemic milieu. The differential transcriptome of hypoxic effects on iPSC-CMs show upregulation of ECM-cell/cell-cell interactions (COL9A1, ITGB6, CTSV, and EPHA1), energy metabolism/hypoxia (ALDOC, ENO2, PFKFB4, CA3, and CA9), and paracrine signaling (WNT, PDGF, FGFR, EGFR, PI3K, and VEGF) in the 3 D format, which suggest engineered heart tissue as more suitable model for evaluating cardiac regenerative therapy.

Keywords: cardiomyocytes; engineered heart tissue; hypoxia; induced pluripotent stem cells; nonhuman primate; transcriptome.

Bronchoalveolar lavage (BAL) has proven to be very useful to monitor the lung allograft after transplantation. In addition to allowing detection of infections, multiple BAL analytes have been proposed as potential biomarkers of lung allograft rejection or dysfunction. However, BAL collection is not well standardized and differences in BAL collection represent an important source of variation. We hypothesized that there are systematic differences between sequential BALs that are relevant to BAL analysis.

As part of 126 consecutive bronchoscopies in lung transplant recipients, two sequential BALs (BAL1 and BAL2) were performed in one location during each bronchoscopy by instilling and suctioning 50 ml of normal saline twice into separate containers. Cell concentration, viability and differentials, Surfactant Protein-D (SP-D), Club Cell Secretory Protein (CCSP), and levels of CXCL10, IL-10, CCL2, CCL5, VEGF-C, RAGE, CXCL9, CXCL1, IL-17A, IL-21, PDGF, and GCSF were compared between BAL1 and BAL2.

Total cell concentration did not differ between BAL1 and BAL2; however, compared to BAL2, BAL1 had more dead cells, epithelial cells, neutrophils, and higher concentrations of airway epithelium-derived CCSP and inflammatory markers. BAL2 had a higher concentration of SP-D compared to BAL1.

In this study performed in lung transplant recipients, we show that sequential BALs represent different lung compartments and have distinct compositions. BAL1 represents the airway compartment with more epithelial cells, neutrophils, and epithelium-derived CCSP. Conversely, BAL2 samples preferentially the distal bronchoalveolar space with greater cell viability and higher SP-D. Our findings illustrate how the method of BAL collection can influence analyte concentrations and further emphasize the need for a standardized approach in translational research involving BAL samples.

Endothelial cell-derived extracellular vesicles (C<em>D</em>31EVs) constitute a new entity for therapeutic/prognostic purposes. The roles of C<em>D</em>31EVs as mediators of vascular smooth muscle cell (VSMC) dysfunction in type 2 diabetes (T2<em>D</em>) are investigated herein. We demonstrated that, unlike serum-derived extracellular vesicles in individuals without diabetes, those in individuals with diabetes (<em>D</em> C<em>D</em>31EVs) boosted apoptosis resistance of VSMCs cultured in hyperglycemic condition. Biochemical analysis revealed that this effect relies on changes in the balance between antiapoptotic and proapoptotic signals: increase of bcl-2 and decrease of bak/bax. <em>D</em> C<em>D</em>31EV cargo analysis demonstrated that <em>D</em> C<em>D</em>31EVs are enriched in membrane-bound platelet-derived growth factor-BB (mb<em>PDGF</em>-BB). Thus, we postulated that mb<em>PDGF</em>-BB transfer by <em>D</em> C<em>D</em>31EVs could account for VSMC resistance to apoptosis. By depleting C<em>D</em>31EVs of platelet-derived growth factor-BB (<em>PDGF</em>-BB) or blocking the <em>PDGF</em> receptor β on VSMCs, we demonstrated that mb<em>PDGF</em>-BB contributes to <em>D</em> C<em>D</em>31EV-mediated bak/bax and bcl-2 levels. Moreover, we found that bak expression is under the control of <em>PDGF</em>-BB-mediated microRNA (miR)-296-5p expression. In fact, while <em>PDGF</em>-BB treatment recapitulated <em>D</em> C<em>D</em>31EV-mediated antiapoptotic program and VSMC resistance to apoptosis, <em>PDGF</em>-BB-depleted C<em>D</em>31EVs failed. <em>D</em> C<em>D</em>31EVs also increased VSMC migration and recruitment to neovessels by means of <em>PDGF</em>-BB. Finally, we found that VSMCs, from human atherosclerotic arteries of individuals with T2<em>D</em>, express low bak/bax and high bcl-2 and miR-296-5p levels. This study identifies the mb<em>PDGF</em>-BB in <em>D</em> C<em>D</em>31EVs as a relevant mediator of diabetes-associated VSMC resistance to apoptosis.

BACKGROUND The aim of this study was to evaluate the effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) on cell proliferation, apoptosis, and the cell cycle in human lung cancer cell lines SK-LU-1, A549, and A-427, and the normal lung fibroblast cell line, MRC-5, in vitro. MATERIAL AND METHODS Human lung adenocarcinoma cells SK-LU-1, A549, and A-427, and the normal lung fibroblast cells, MRC-5 were cultured and treated with increasing concentrations of 10 mM of a stock solution of THOH in dimethyl sulfoxide (DMSO). An MTT cell proliferation assay was used. Cell apoptosis and the cell cycle were studied using fluorescence-activated cell sorting (FACs) with fluorescein isothiocyanate (FITC), Annexin-V, propidium iodide (PI), and nuclear staining with 4',6-diamidino-2-phenylindole (DAPI). DNA damage was measured using the comet (single-cell gel electrophoresis) assay. Cell migration was evaluated using a wound healing assay, and Western blotting was used to measure protein expression levels. RESULTS Treatment of SK-LU-1 cells with THOH inhibited cell migration. Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, with the most marked inhibition found in the SK-LU-1 lung cancer cells (IC50, 12 µM). Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH increased cell apoptosis, resulted in G2/M cell cycle arrest, and inhibited both the platelet-derived growth factor D (PDGF-D) and MEK/ERK signaling pathways. CONCLUSIONS Treatment of adenocarcinoma cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, apoptosis, and resulted in G2/M cell cycle arrest by targeting PDGF-D and the MEK/ERK signaling pathway.

Current methods for articular cartilage repair are unpredictable with respect to clinical success. In the present study, we investigated the ability of cells from articular cartilage, perichondrium, and costochondral resting zone to form new cartilage when loaded onto biodegradable scaffolds and implanted into calf muscle pouches of nu/nu mice. Prior in vitro studies showed that platelet derived growth factor-BB (PDGF-BB), but not transforming growth factor beta-1 (TGF-beta 1), basic fibroblast growth factor, or bone morphogenetic protein-2 promoted proliferation and extracellular matrix sulfation of resting zone chondrocytes without causing the cells to exhibit a hypertrophic chondrocyte phenotype. TGF-beta 1 has also been shown to stimulate chondrogenesis by multipotent chondroprogenitor cells like those in the perichondrium. In addition, PDGF-BB has been shown to modulate chondrogensis by resting zone cells implanted in poly(D,L-lactide-co-glycolide) (PLG) scaffolds. In the present study we examined whether the cartilage formation is dependent on state of chondrocyte maturation and whether the pretreatment of chondrocytes with growth factors has an influence on the cartilage formation. Scaffolds were manufactured from 80% PLG with a 75:25 lactide:glycolide ratio and 20% modified PLG with a 50:50 lactide:glycolide ratio (PLG-H scaffolds). For each experimental group, four nude mice received two identical implants, one in each calf muscle resulting in an N = 8 implants: PLG-H scaffolds alone; PLG-H scaffolds with cells derived from either the femoral articular cartilage, costochondral periochondrium, or costochondral resting zone cartilage of 125 g male Sprague-Dawley rats; PLG-H scaffolds with either articular chondrocytes or resting zone chondrocytes that were pretreated with 37.5 ng/ml rhPDGF-BB for 4 h or 24 h before implantation, or with perichondrial cells treated with PDGF-BB plus 0.22 ng/ml rhTGF beta-1 for 4 h and 24 h. At 4 or 8 weeks after implantation, samples were harvested and analyzed histomorphometrically for new cartilage formed, area of residual implant and area of fibrous connective tissue. Only resting zone cells showed the ability to form new cartilage at a heterotopic site in this study. There was no neocartilage found in nude mice with implants loaded with either articular chondrocytes or perichondrial cells. Pretreatment of resting zone chondrocytes for 4 h prior to implantation significantly increased the amount of newly formed cartilage after 8 weeks and suppressed chondrocyte hypertrophy. The amount of fibrous connective tissue around implants containing either articular chondrocytes or perichondrial cells decreased with time, whereas the amount of fibrous connective tissue around implants containing resting zone chondrocytes pretreated with PDGF-BB was increased. The results showed that resting zone cells can be successfully incorporated into biodegradable porous PLG scaffolds and can induce new cartilage formation in a nonweight-bearing site. Articular chondrocytes as well as perichondrial cells did not have the capacity for neochondrogenesis when implanted heterotopically in this model.

Horses are more prone to complications in the wound healing process than other species, and problems such as chronic inflammation, delayed epithelialization, poor wound contraction, and exuberant granulation tissue are commonly seen, particularly in wounds on the distal limbs. In comparison, wounds of the oral mucosa heal rapidly in a scarless fashion with a high degree of wound contraction. The effect of platelet-derived growth factor BB (PDGF), insulin-like growth factor (IGF)-1, and transforming growth factor beta1 (TGFbeta1) on the contraction of a fibroblast-populated collagen matrix (FPCM) as a model of equine wound contraction was investigated using equine oral fibroblasts. The fibroblasts were embedded into floating FPCM and treated with PDGF, IGF-1, and TGFbeta1. The surface areas of the FPCM were determined daily for 5 d. Platelet-derived growth factor significantly stimulated the contraction of the FPCM at an optimal concentration of 10 ng/mL (P=0.025). Insulin-like growth factor-1 and TGFbeta1 did not significantly affect the contraction of the FPCM relative to the control. To elucidate the mechanisms by which PDGF stimulated contraction of FPCM, the Rho-kinase and p38 cell signaling pathways were blocked, resulting in a significant inhibition (P<0.001) of PDGF-stimulated contraction. Platelet-derived growth factor BB is a potent stimulator of fibroblast migration, and hence the FPCM contraction generated here is probably a result of its effects on cell migration. The results of the present experiment suggest that this effect is stimulated via both the Rho-kinase and p38 signaling pathways in equine oral fibroblasts.

OBJECTIVE

To investigate the method of differentiating dental pulp stem cells (DPSCs) modified by HIF-1α into blood vessels.

METHODS

DPSCs were extracted from teeth samples from 20 patients and were identified by Strol-1 and CD146. DPSCs were divided into experimental group and control group according to DPSCs were modified by HIF-1α not or. HIF-1α-mRNA expression was detected by RT-PCR. HIF-1α, VEGF, SDF-1, Ang-2 and PDGF expression were detected using Western blot in different time after culture for 1 d, 4 d, 7 d and 14 d. Statistical analysis was carried out with SPSS 16.0 software package.

RESULTS

Most DPSCs appeared round, oval under phase-contrast microscopy. CD146 and Strol-1 showed green fluorescence. HIF-1α and HIF-1α-mRNA expression became higher with time passing and the difference was statistically significant (P<0.05). Compared with the control group, HIF-1α protein and mRNA increased obviously in the experimental group 1d, 4d, 7d and 14d after transfection, and the difference was statistically significant (P<0.05). The level of VEGF, SDF-1, Ang-2 and PDGF in the control group was changed unconspicuously, and the expression was not different at different times (P>0.05). The level of VEGF, SDF-1, Ang-2 and PDGF in the exprimental group increased, and the difference was statistically significant between different time points(P<0.05). Compared with the control group, the level of VEGF, SDF-1, Ang-2 and PDGF in the experimental group was higher 1 d, 4 d, 7 d and 14 d after transfection, respectively, and the difference was statistically significant(P<0.05).

CONCLUSIONS

DPSCs modified by HIF-1α gene can successfully induce vascular differentiation in vitro, which provides foundation for further angioplasty.

To determine whether cytomegalovirus (CMV) infection alters growth factor production from endothelial cells (EC) or fibroblasts, we infected human umbilical vein EC with CMV VHL/E, a strain of CMV with affinity for human EC, and we infected human foreskin fibroblasts with CMV AD169. CMV caused cytopathic effect and positive CMV staining by immunofluorescence within 5 d, effects not seen in cells infected with UV-irradiated CMV or in uninfected (control) cells. The supernatants from the EC were assayed for platelet-derived growth factor (PDGF)-like protein using a radioreceptor inhibition assay, and EC and fibroblasts were assayed for basic fibroblast growth factor (bFGF) by Western blot analysis. There were no significant differences in PDGF production between groups of EC: CMV-infected EC, 13.5 +/- 2.6; UV-irradiated infected EC, 12.1 +/- 3.6; control EC, 12.9 +/- 1.7 fmol/10(6) EC (mean +/- SD, n = 10, p = NS). There were also no significant differences in bFGF production between CMV-infected EC, UV-irradiated infected EC, and control EC as evidenced by similar intensity of migration of bFGF as a single band at approximately 18 kD (n = 5). In contrast, CMV infection of fibroblasts induced a shift in production of bFGF to higher molecular weight forms migrating at 24 and 26 kD molecular mass. alpha-Interferon failed to alter bFGF production. We conclude that CMV VHL/E infection of EC does not directly alter PDGF or bFGF production from EC. However, CMV infection of cultured human fibroblasts qualitatively alters bFGF by inducing a shift to higher molecular weight forms.

Real time protein signaling in a complex medium may provide a promising way for high-throughput protein analysis, but it is largely unmet due to the challenge of signal transduction and the interferences of nonspecific binding and high background. Our recent work indicates that a fluorescent aptamer can display a protein binding-induced reduction of fluorescence anisotropy (FA) (Zhang, D.; Lu, M.; Wang, H. J. Am. Chem. Soc. 2011, 133, 9188-9191), which is exclusively different from a traditionally simplified concept hinting a molecular size increase-induced FA increase. Inspired by this unexpected observation, we describe a novel FA reduction approach for protein signaling. The feasibility of this approach is demonstrated through the assays of a blood protein human α-thrombin and an oncoprotein human platelet-derived growth factor B-chain (PDGF-BB) using two screened fluorescent aptamers, respectively. By the developed FA reduction method, the spiked human α-thrombin in diluted serum can be detected at the concentration as low as 250 pM. In contrast, in a traditional molecular size-dependent FA assay, the thrombin spiked in diluted serum cannot induce reliable FA change even at a 256-fold higher concentration (64 nM). The results clearly show that the FA reduction approach has a dramatically enhanced specificity against target protein and high sensitivity in complex medium and is applicable to the no-separation based detection of proteins in biological matrixes.