Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRβ+ CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11-deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin α11 and PDGFRβ was found at both transcriptional and histological levels in BC specimens. High stromal integrin α11/PDGFRβ expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using five CAF subpopulations (one murine, four human) revealed that integrin α11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, integrin α11 pro-invasive activity relies on its ability to interact with PDGFRβ in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a pro-invasive matricellular protein. Pharmacological inhibition of PDGFRβ and JNK impaired tumor cell invasion induced by integrin α11-positive CAFs. Collectively, our study uncovers an integrin α11-positive subset of pro-tumoral CAFs that exploits PDGFRβ/JNK signalling axis to promote tumor invasiveness in BC.

BACKGROUND

Platelet-derived growth factor (PDGF) signalling is essential for many key cellular processes in mesenchymal cells. As there is redundancy in signalling between the five PDGF ligand isoforms and three PDGF receptor isoforms, and deletion of either of the receptors in vivo produces an embryonic lethal phenotype, it is not know which ligand and receptor combinations mediate specific cellular functions. Fibroblasts are key mediators in wound healing and tissues repair. Recent clinical trials using broad spectrum tyrosine kinase inhibitors in fibrotic diseases have highlighted the need to further examine the specific cellular roles each of the tyrosine kinases plays in fibrotic processes. In this study, we used PDGFR-specific neutralising antibodies to dissect out receptor-specific signalling events in fibroblasts in vitro, to further understand key cellular processes involved in wound healing and tissue repair.

RESULTS

Neutralising antibodies against PDGFRs were shown to block signalling through PDGFRα and PDGFRβ receptors, reduce human PDGF-AA and PDGF-BB-induced collagen gel remodelling in dermal fibroblasts, and reduce migration stimulated by all PDGF ligands in human dermal and lung fibroblasts.

CONCLUSIONS

PDGFRα and PDGFRβ neutralising antibodies can be a useful tool in studying PDGFR isoform-specific cellular events.

BACKGROUND

Intimal hyperplasia is a common cause of many vasculopathies. There has been a recent surge of interest in the bromo and extra-terminal (BET) epigenetic "readers" including BRD4 since the serendipitous discovery of JQ1(+), an inhibitor specific to the seemingly undruggable BET bromodomains. The role of the BET family in the development of intimal hyperplasia is not known.

METHODS

We investigated the effect of BET inhibition on intimal hyperplasia using a rat balloon angioplasty model.

RESULTS

While BRD4 was dramatically up-regulated in the rat and human hyperplastic neointima, blocking BET bromodomains with JQ1(+) diminished neointima in rats. Knocking down BRD4 with siRNA, or treatment with JQ1(+) but not the inactive enantiomer JQ1(-), abrogated platelet-derived growth factor (PDGF-BB)-stimulated proliferation and migration of primary rat aortic smooth muscle cells. This inhibitory effect of JQ1(+) was reproducible in primary human aortic smooth muscle cells. In human aortic endothelial cells, JQ1(+) prevented cytokine-induced apoptosis and impairment of cell migration. Furthermore, either BRD4 siRNA or JQ1(+) but not JQ1(-), substantially down-regulated PDGF receptor-α which, in JQ1(+)-treated arteries versus vehicle control, was also reduced.

CONCLUSIONS

Blocking BET bromodomains mitigates neointima formation, suggesting an epigenetic approach for effective prevention of intimal hyperplasia and associated vascular diseases.

OBJECTIVE

We investigated the regulation of p38 mitogen-activated protein kinase (MAPK) by platelet-derived growth factor (PDGF)-BB and its biological effects in cultured normal and diabetic rat vascular smooth muscle cells (VSMCs).

RESULTS

VSMC growth from diabetic rats was faster than that from normal rats. The expression of the PDGF beta-receptor in diabetic VSMCs was significantly elevated compared with that in normal cells, and PDGF-BB-induced p38 phosphorylation in diabetic cells was more enhanced via MAPK kinase (MKK) 3/6. The level of PKC activity in diabetic cells increased more than that in normal cells with or without PDGF-BB. Although protein kinase C (PKC)-betaII and PKC-delta were activated by diabetes, PDGF-BB could further enhance the level of PKC-delta alone. PDGF-BB-induced cell migration was more elevated in diabetic VSMCs, and the increase was significantly inhibited by SB-203580, rottlerin, and antisense oligodeoxynucleotides for PKC-delta. PDGF-BB-induced p38 phosphorylation also regulated cell growth, cyclooxygenase-2 levels, and arachidonic acid release, but not apoptosis. These levels were more elevated in diabetic cells, which were inhibited by SB-203580.

CONCLUSIONS

Our study established that PDGF-BB phosphorylated p38 via PKC-delta and the subsequent MKK 3/6, leading to cell growth regulation and the progression of a chronic inflammatory process in diabetic VSMCs.

BACKGROUND

After endovascular injury, smooth muscle cells (SMCs) may be exposed to hemodynamic shear stress (SS), and these forces modulate neointima accumulation. The effect of SS on SMC migration and invasion is unknown, and it was examined in the present study.

RESULTS

Bovine aortic SMCs were exposed to laminar SS of 12 dyne/cm(2) for 3 (SS3) or 15 (SS15) hours; control (C3 and C15) SMCs were kept under static conditions. Platelet-derived growth factor (PDGF)-BB-directed SMC migration and invasion were evaluated by a modified Boyden chamber assay with filters coated with either gelatin or reconstituted basement membrane proteins (Matrigel), respectively. SS15 inhibited both SMC migration and invasion (P<0.0001). There was no significant difference between SS3 and C3 cells. Media conditioned with SS15 cells exhibited a reduction in matrix metalloprotease-2 (MMP-2) by zymography and Western analysis. Northern blot analysis revealed no effect of SS15 on MMP-2 mRNA. In contrast, SS15 decreased MMP-2 activator and membrane-type MMP (MT-MMP or MMP-14) mRNA and protein. Furthermore, SS15 decreased PDGF receptor-beta (PDGF-Rbeta) mRNA and protein (P<0.05), and the SS-dependent decrease in PDGF-BB-directed cell migration was rescued by overexpressing PDGF-Rbeta.

CONCLUSIONS

SS inhibits SMC migration and invasion via diminished PDGF-Rbeta expression. This effect of SS is associated with decreased MMP-2 secretion and MT-MMP downregulation.

OBJECTIVE

Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease.

RESULTS

Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [3H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (P<0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation.

CONCLUSIONS

Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2.

Despite intensive research studies, theories have yet to focus on the contribution of hypoxia to patency differences observed clinically between arterial vs. venous grafts. This study investigates the differential hypoxic response of smooth muscle cells (SMC) to hypoxia-derived endothelial cell (EC) growth factors. Initiation of SMC proliferation under hypoxia (<5% O(2)) occurred only after incubation with hypoxic endothelial cell-conditioned media (H-ECM). After the investigation of several possible growth factors in the H-ECM that may be responsible for SMC proliferation, the greatest difference was observed in vascular endothelial growth factor (VEGF-A) and platelet-derived growth factor homodimer B (PDGF-BB) expression. VEGF-A increased (2-fold) significantly (P < 0.05) in arterial-derived smooth muscle cells (ASMC) under hypoxia compared with venous-derived smooth muscle cells (VSMC), which showed no significant change. VSMC showed significant (P < 0.05) increase in VEGFR-2 expression under hypoxia compared with ASMC. Incubation with VEGFR-2-neutralizing antibody/PDGFR antagonist in VSMC before addition of H-ECM resulted in decreased proliferation. ASMC proliferation under hypoxia did not decrease during incubation with VEGFR-2-neutralizing antibody but did decrease upon PDGFR antagonist incubation. Current therapies focusing on treating intimal hyperplasia have negated the fact that combinational therapy might be required to combat induction of SMC proliferation. Clinically, therapy with PDGFR antagonists plus anti-VEGFR-2 may prove to be efficacious in managing SMC proliferation in venous-derived grafts.

The urgent needs of functional arterial replacements for curing the vascular system diseases have been proposed for many years. However, an ideal small-diameter vascular scaffold, which is nonthrombogenic, minimizes intimal hyperplasia, matches the mechanical properties of natural vessels, and supports neovascular tissue reconstruction, is still in progress. For this purpose, we previously attempted dual-delivery of VEGF and PDGF by double-layered electrospun membranes. Here, a multilayered vascular scaffold in 1.5-mm diameter with sufficient mechanical properties was developed by electrospinning from poly(ethylene glycol)-b-poly(L-lactide-co-ε-caprolactone) (PELCL), poly(L-lactide-co-glycolide) (PLGA), poly(ε-caprolactone) (PCL) and gelatin. Spatio-temporal releases of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGF) were specially controlled by the inner PELCL and middle PLGA layers, respectively, and the outer PCL layer contributed to the mechanical stability. Introduction of gelatin improved vascular endothelial cells adhesion at first, and loosen membrane after its degradation facilitated vascular smooth muscle cells (VSMCs) ingrowth. Cell activities indicated dual release of growth factors promoted endothelialization and inhibited VSMCs hyperproliferation. The small-diameter vascular scaffold dual-loading VEGF and PDGF could maintain patency in rabbit left common carotid artery for 8 weeks. It is concluded that the specially prepared fibrous scaffold in multilayer could benefit blood vessel reconstruction.

Spaceflight is known to diminish bone mass and reduce immune function, suggesting that repair of connective tissue might be impaired in a microgravity environment. Fisher 344 rats were used to test wound healing responses in the orbiting Space Shuttle Endeavour by preflight implantation of polyvinyl acetal sponge disks in which pellets were placed to release either platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF), or placebo. Control groups on the ground included a matched environment group in similar housing modules and temperature control groups in cages at 22 degreesC and 28 degreesC. After 12 days of implantation and 10 days in orbit, the removed sponges were analyzed for histological and biochemical responses. Growth factor responses were histologically evident after release of PDGF-BB and bFGF in ground controls, whereas only immediate-release bFGF and delayed-release PDGF-BB showed significant responses in microgravity. Biochemical data confirmed that cellularity was increased by both factors in ground sponges; however, this response was significantly blunted in flight sponges (P<0.005, ANOVA), irrespective timing of factor release. Collagen content was 62% lower in sponges from animals with 10 days of microgravity exposure (P<0.01, ANOVA) and further reduced by bFGF. These data suggest that orbital exposure retards the capacity of wounds to heal and respond to exogenous stimuli.

Background: Aberrant expression of circular RNA (CircRNA) contributes to human diseases. CircRNAs regulate gene expression by sequestering specific microRNAs (miRNAs). In this study, we investigated whether CircMAP3K5 could act as a competing endogenous miR-22-3p sponge and regulate neointimal hyperplasia. Methods: CircRNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (HCASMCs) treated with or without PDGF. Expression levels of circular MAP3K5 (CircMAP3K5) was assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy (DCM) or coronary heart disease (CHD). The role of CircMAP3K5 in intimal hyperplasia was further investigated in mice with AAV9-mediated CircMAP3K5 transfection. SMC-specific Tet2 knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which CircMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. Results: RNA sequencing demonstrated that treatment with PDGF-BB significantly reduced expression of CircMAP3K5 in HCASMCs. Wire-injured mouse femoral arteries and diseased arteries from CHD patients (where PDGF-BB is increased) confirmed in vivo downregulation of CircMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of CircMAP3K5 inhibited the proliferation of HCASMCs. In vivo AAV9-mediated transfection of CircMap3k5 specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, CircMAP3K5 was found to sequester miR-22-3p, which in turn inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the anti-proliferative effect of CircMap3k5 on VSMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the CircMap3k5-mediated anti-proliferative effect on VSMCs. Conclusions: We identify CircMAP3K5 as a master regulator of TET2-mediated VSMC differentiation. Targeting the CircMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia including restenosis and atherosclerosis.

Keywords: Circular RNA CircMAP3K5; MicroRNA-22-3p; TET2; intimal hyperplasia.

Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) beta-receptor (PDGFRbeta) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRbeta removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRbeta. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRbeta. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRbeta. Our data suggest that Alix inhibits down-regulation of PDGFRbeta by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.

BACKGROUND

Obesity is associated with numerous health complications; however, a subgroup of obese individuals (termed the metabolically healthy obese or MHO) appear to have lower risk for complications such as type 2 diabetes and cardiovascular disease. Emerging evidence suggests that MHO individuals have reduced inflammation compared to their metabolically unhealthy obese (MUO) counterparts. As it is recognized that fatty acids (FAs) have a strong relationship with inflammation, the current study aimed to uncover if the reduced inflammation observed in MHO individuals is mirrored by a more favourable FA profile.

METHODS

Fasted serum samples were collected from lean healthy (LH), MHO, and MUO participants (n = 10/group) recruited from the Diabetes Risk Assessment study. A panel of pro- and anti-inflammatory markers were measured by immunoassay. Total serum FA profiling, as well as the FA composition of circulating phospholipids (PL) and triglycerides (TG), was measured by gas chromatography. ANOVA and Mann-Whitney-Wilcoxon tests were used to assess statistical significance between the groups (P<0.05).

RESULTS

MHO and MUO individuals had similar BMI and body fat %; however, lipid parameters in MHO individuals more closely resembled that of LH individuals. MHO individuals had circulating levels of high sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) similar to LH individuals, while levels of platelet derived growth factor-ββ (PDGF-ββ) were intermediate to that of LH and MUO individuals. FA profiling analysis combined with discriminant analysis modelling highlighted a panel of nine FAs (consisting of three saturated, three monounsaturated, and three polyunsaturated FAs) in PL and TG fractions that distinguished the three groups. Specifically, saturated FA (myristic and stearic acids) levels in MHO individuals resembled that of LH individuals.

CONCLUSIONS

Our results suggest that the reduced inflammatory state of MHO individuals compared to MUO individuals may stem, in part, from a more favourable underlying FA profile.

Platelet-derived growth factor (PDGF) receptor signaling plays an important role in the regulation of proliferation and migration of skeletal cells such as osteoblasts or mesenchymal stem cells (MSCs). However, involvement of these receptors in the process of osteoblastic differentiation of MSCs is still a matter of debate. The aim of our study was to examine the role of PDGF receptor signaling in osteogenic differentiation of human MSCs. For this purpose, we performed PDGF receptor stimulation as well as inhibition experiments. Inhibition experiments were carried out with Tyrphostin AG1296, a potent and specific inhibitor of PDGF receptor activity. As expected, Tyrphostin AG1296 treatment caused a concentration-dependent decrease in fetal calf serum and PDGF-BB-induced proliferation of MSCs and effectively inhibited PDGF-BB-induced phosphorylation of extracellular-regulated kinase 1/2. However, PDGF receptor inhibition had no significant effect on osteoblastic differentiation of MSCs, as evaluated histochemically by von Kossa, Alizarin-Red, and osteocalcin stainings. Moreover, mineralized matrix production, as assayed by quantitative Ca(2+)-measurements, was also not modulated by Tyrphostin AG1296 treatment. These results were noticeable irrespective of whether MSCs were grown under nonosteogenic or osteogenic differentiation conditions. Similarly, PDGF-BB treatment of MSCs in receptor stimulation experiments also failed to modulate mineralization. However, expression of alkaline phosphatase was suppressed by Tyrphostin AG1296 treatment at later stages of osteogenesis but not in the early stages, as assessed by enzyme activity and mRNA expression assays. Expression of other osteogenic marker genes such as osteocalcin, runt-related transcription factor 2, osteopontin, collagen type I, and bone sialoprotein was almost unaffected in our perturbation studies. From these experiments, we conclude that PDGF receptor signaling sustains proliferation without affecting osteogenic differentiation of MSCs.

Phenotypic switch of vascular smooth muscle cells (VSMCs) is characterized by increased expressions of VSMC synthetic markers and decreased levels of VSMC contractile markers, which is an important step for VSMC proliferation and migration during the development and progression of cardiovascular diseases including atherosclerosis. Chicoric acid (CA) is identified to exert powerful cardiovascular protective effects. However, little is known about the effects of CA on VSMC biology. Herein, in cultured VSMCs, we showed that pretreatment with CA dose-dependently suppressed platelet-derived growth factor type BB (PDGF-BB)-induced VSMC phenotypic alteration, proliferation and migration. Mechanistically, PDGF-BB-treated VSMCs exhibited higher mammalian target of rapamycin (mTOR) and P70S6K phosphorylation, which was attenuated by CA pretreatment, diphenyleneiodonium chloride (DPI), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) and nuclear factor-κB (NFκB) inhibitor Bay117082. PDGF-BB-triggered ROS production and p65-NFκB activation were inhibited by CA. In addition, both NAC and DPI abolished PDGF-BB-evoked p65-NFκB nuclear translocation, phosphorylation and degradation of Inhibitor κBα (IκBα). Of note, blockade of ROS/NFκB/mTOR/P70S6K signaling cascade prevented PDGF-BB-evoked VSMC phenotypic transformation, proliferation and migration. CA treatment prevented intimal hyperplasia and vascular remodeling in rat models of carotid artery ligation in vivo. These results suggest that CA impedes PDGF-BB-induced VSMC phenotypic switching, proliferation, migration and neointima formation via inhibition of ROS/NFκB/mTOR/P70S6K signaling cascade.

During platelet-derived growth factor (PDGF)-BB-mediated recruitment to neovascular sprouts, vascular smooth muscle cells (VSMCs) dedifferentiate from a contractile to a migratory phenotype. This involves the downregulation of contractile markers such as smooth muscle (SM) alpha-actin and the upregulation of promigration genes such as matrix metalloproteinase (MMP)-2. The regulation of MMP-2 in response to PDGF-BB is complex and involves both stimulatory and inhibitory signaling pathways, resulting in a significant delay in upregulation. Here, we provide evidence that the delay in MMP-2 upregulation may be due to the autocrine expression and activation of transforming growth factor (TGF)-beta, which is known to promote the contractile phenotype in VSMCs. Whereas PDGF-BB could induce the loss of stress fibers and focal adhesions, TGF-beta was able to block or reverse this transition to a noncontractile state. TGF-beta did not, however, suppress early signaling events stimulated by PDGF-BB. Over time, though PDGF-BB induced increased TGF-beta1 levels, it suppressed TGF-beta2 and TGF-beta3 expression, leading to a net decrease in the total TGF-beta pool, resulting in the upregulation of MMP-2. Together, these findings indicate that MMP-2 expression is suppressed by a threshold level of active TGF-beta, which in turn promotes a contractile VSMC phenotype that prevents the upregulation of MMP-2.

Cancer-associated fibroblasts (CAFs) represent the predominant cell type of the neoplastic stroma of solid tumors, yet their biology and functional specificity for cancer pathogenesis remain unclear. We show here that primary CAFs from colorectal liver metastases express several inflammatory, tumor-enhancing factors, including interleukin (IL)-6 and monocyte-chemoattractant protein (MCP)-1. Both molecules were intensely induced by TNF-alpha on the transcript and protein level, whereas PDGF-BB, TGF-beta1 and EGF showed no significant effects. To verify their potential specialization for metastasis progression, CAFs were compared to fibroblasts from non-tumor liver tissue. Interestingly, these liver fibroblasts (LFs) displayed similar functions. Further analyses revealed a comparable up-regulation of intercellular adhesion molecule-1 (ICAM-1) by TNF-alpha, and of alpha-smooth muscle actin, by TGF-beta1. Moreover, the proliferation of both cell types was induced by PDGF-BB, and CAFs and LFs displayed an equivalent migration towards HT29 colon cancer cells in Boyden chamber assays. In conclusion, colorectal liver metastasis may be supported by CAFs and resident fibroblastic cells competent to generate a prometastatic microenvironment through inflammatory activation of IL-6 and MCP-1.

Vascular smooth muscle cells (SMCs) occur throughout the vascular tree and have important physiological functions. They are also involved in pathological processes such as development and progression of atherosclerotic lesions, restenosis following angioplasty, and in hypertension. This review is focused on the role of the insulin-like growth factor (IGF) system in proliferation, migration, and hypertrophy of vascular SMCs and its interaction with insulin and other growth factors. The IGF-I receptor is highly expressed in SMCs in intact arteries and in cultured SMCs and is activated by binding of IGF-I to the two alpha-subunits. Insulin and IGF-II from the circulation can interact with the IGF-I receptor at higher concentrations. Insulin receptors are few or absent in SMCs and circulating insulin concentrations in vivo are probably too low for a direct action of insulin on the IGF-I receptor in SMCs. Receptor activation initiates a number of signal transduction pathways. Increased phosphatidylinositol turnover and calcium mobilization correlates with actin filament reorganization and stimulation of directed migration of the SMC in a gradient of IGF-I. The effects of IGF-I receptor activation on signal transduction pathways (eg, the MAP kinase cascade) implicated in DNA synthesis and proliferation are weak and this correlates with the meager mitogenic activity of IGF-I in SMC. Several components of the IGF-system in SMC are regulated by growth factors such as platelet-derived growth factor (PDGF)-BB and basic fibroblast growth factor (bFGF).(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) modulate vascular wall cell function in vitro and angiogenesis in vivo. The aim of the current study was to determine how bovine aorta endothelial cells (BAECs) respond to the simultaneous exposure to PDGF-BB and bFGF. It was found that bFGF-dependent BAEC migration, proliferation, and differentiation into tubelike structures on reconstituted extracellular matrix (Matrigel) were inhibited by PDGF-BB. The role played by PDGF receptor alpha (PDGF-Ralpha) was investigated by selective stimulation with PDGF-AA, by blocking PDGF-BB-binding to PDGF-Ralpha with neomycin, or by transfecting cells with dominant-negative forms of the receptors to selectively impair either PDGF-Ralpha or PDGF-Rbeta function. In all cases, PDGF-Ralpha impairment abolished the inhibitory effect of PDGF-BB on bFGF-directed BAEC migration. In addition, PDGF-Ralpha phosphorylation was increased in the presence of bFGF and PDGF, as compared to PDGF alone, whereas mitogen-activated protein kinase phosphorylation was decreased in the presence of PDGF-BB and bFGF compared with bFGF alone. In vivo experiments showed that PDGF-BB and PDGF-AA inhibited bFGF-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and that PDGF-BB inhibited bFGF-induced angiogenesis in Matrigel plugs injected subcutaneously in CD1 mice. Taken together these results show that PDGF inhibits the angiogenic properties of bFGF in vitro and in vivo, likely through PDGF-Ralpha stimulation.

Coronary vessels develop from a primary vascular network that differentiates in the subepicardium through a process of vasculogenesis, that is, self-assembly of mesenchymal vascular progenitors. Further growth of the subepicardial vascular plexus through a complex process of angiogenesis, vascular remodeling, and arterialization of specific branches gives rise to the definitive coronary system. This report is intended to summarize current knowledge on the origin of the coronary vascular progenitors and to provide new insights suggested by recent findings. It has been established that the mesenchymal precursors of the vascular smooth muscle cells and the adventitial fibroblasts originate from an epithelial-mesenchymal transformation of the epicardial mesothelium. We report herein experimental evidence that the precursors of the coronary endothelium are also epicardium-derived cells (EPDCs). The evidence shown includes co-localization of mesothelial and endothelial molecular markers as well as cell lineage studies performed through direct labeling of the epicardial cells. If this proposal is confirmed, the early EPDCs might be found to have a competence similar to that shown by the recently discovered bipotential vascular progenitor cells, which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDCs differentiate into endothelial cells in response to myocardially secreted VEGF, while subsequent EPDCs, recruited by the nascent capillaries via PDGFRbeta signaling, differentiate into percytes and smooth muscle cells.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) plays an important role in vascular diseases. The Rho-associated protein kinase (ROCK) signaling pathway is now well recognized for its role in VSMC migration and proliferation. Recently, a number of studies revealed that different isoforms of ROCK have distinct functions in VSMCs. We have reported that ROCK1, rather than ROCK2, induces platelet-derived growth factor (PDGF)-BB-stimulated migration of VSMCs. In the current study, we aimed to investigate the roles of ROCK1/2 in PDGF-induced rat aorta VSMC proliferation by manipulating ROCK gene expression. The results revealed that knock-down of both ROCK1 and ROCK2 by siRNA technology decreased PDGF-BB-generated VSMC proliferation by inhibiting the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1. In addition, up-regulation of ROCK1 expression through transfection, further increased the proliferation of VSMCs induced by PDGF-BB. The ERK inhibitor U0126 reduced the proliferation and expression of PCNA and cyclinD1, and ROCK1 and ROCK2 siRNA decreased the level of ERK in the nucleus. These results demonstrated that ROCK1 and ROCK2 could promote VSMC proliferation through ERK nuclear translocation, regulating the expression of PCNA and cyclin D1 protein.

OBJECTIVE

It is well-established that exercise diminishes cardiovascular risk, but whether humoral factors secreted by muscle confer these benefits has not been conclusively shown. We have shown that the secreted protein follistatin-like 1 (Fstl1) has beneficial actions on cardiac and endothelial function. However, the role of muscle-derived Fstl1 in proliferative vascular disease remains largely unknown. Here, we investigated whether muscle-derived Fstl1 modulates vascular remodelling in response to injury.

RESULTS

The targeted ablation of Fstl1 in muscle led to an increase in neointimal formation following wire-induced arterial injury compared with control mice. Conversely, muscle-specific Fstl1 transgenic (TG) mice displayed a decrease in the neointimal thickening following arterial injury. Muscle-specific Fstl1 ablation and overexpression increased and decreased, respectively, the frequency of BrdU-positive proliferating cells in injured vessels. In cultured human aortic smooth muscle cells (HASMCs), treatment with human FSTL1 protein decreased proliferation and migration induced by stimulation with PDGF-BB. Treatment with FSTL1 enhanced AMPK phosphorylation, and inhibition of AMPK abrogated the inhibitory actions of FSTL1 on HASMC responses to PDGF-BB. The injured arteries of Fstl1-TG mice exhibited an increase in AMPK phosphorylation, and administration of AMPK inhibitor reversed the anti-proliferative actions of Fstl1 on the vessel wall.

CONCLUSIONS

Our findings indicate that muscle-derived Fstl1 attenuates neointimal formation in response to arterial injury by suppressing SMC proliferation through an AMPK-dependent mechanism. Thus, the release of protein factors from muscle, such as Fstl1, may partly explain why the maintenance of muscle function can have a therapeutic effect on the cardiovascular system.

We devised and tested an in vivo system to monitor the migration of mesenchymal stem cells (MSCs) within the marrow cavity. In vitro studies confirmed that platelet-derived growth factor (PDGF)-AA had the most potent chemotactic effect of the tested factors, and possessed the greatest number of receptors in MSCs. MSCs were labeled with fluorescent nanoparticles and injected into the marrow cavity of nude rats through osteochondral defects created in the distal femur. The defects were sealed with HCF (heparin-conjugated fibrin) or PDGF-AA-loaded HCF. In the HCF-only group, the nanoparticle-labeled MSCs dispersed outside the marrow cavity within 3 days after injection. In the PDGF-AA-loaded HCF group, the labeled cells moved time-dependently for 14 days toward the osteochondral defect. HCF-PDGF in low dose (LD; 8.5 ng/µl) was more effective than HCF-PDGF in high dose (HD; 17 ng/µl) in recruiting the MSCs to the osteochondral defect. After 21 days, the defects treated with PDGF and transforming growth factor (TGF)-β1-loaded HCF showed excellent cartilage repair compared with other groups. Further studies confirmed that this in vivo osteochondral MSCs tracking system (IOMTS) worked for other chemoattractants (chemokine (C-C motif) ligand 2 (CCL2) and PDGF-BB). IOMTS can provide a useful tool to examine the effect of growth factors or chemokines on endogenous cartilage repair.

The epicardium develops from an extracardiac primordium, the proepicardium, which is constituted by a cluster of mesothelial cells located on the cephalic and ventral surface of the liver-sinus venosus limit (avian embryos) or on the pericardial side of the septum transversum (mammalian embryos). The proepicardium contacts the myocardial surface and gives rise to a mesothelium, which grows and progressively lines the myocardium. The epicardium generates, through a process of epithelial-mesenchymal transition, a population of epicardial-derived cells (EPDC). EPDC contribute to the development of cardiac connective tissue, fibroblasts, and the smooth muscle of cardiac vessels. Recent data suggest that EPDC can also differentiate into endothelial cells of the primary subepicardial vascular plexus. If this is confirmed, EPDC would show the same developmental properties that characterize the stem-cell-derived bipotential vascular progenitors recently described, whose differentiation into endothelium and smooth muscle is regulated by exposure to VEGF and PDGF-BB, respectively. Aside from their function in the development of cardiac connective and vascular tissue, EPDC also play an essential modulating role in the differentiation of the compact ventricular layer of the myocardium, a role which might be regulated by the transcription factor WT1 and the production of retinoic acid.

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.