Loss of microRNA-17∼92 in smooth muscle cells attenuates experimental pulmonary hypertension via induction of PDZ and LIM domain 5.

RATIONALE: Recent studies suggest that microRNAs (miRNAs) play important roles in regulation of pulmonary artery smooth muscle cell (PASMC) phenotype and are implicated in pulmonary arterial hypertension (PAH). However, the underlying molecular mechanisms remain elusive. OBJECTIVES: This study aims to understand the mechanisms regulating PASMC proliferation and differentiation by microRNA-17∼92 (miR-17∼92) and to elucidate its implication in PAH. METHODS: We generated smooth muscle cell (SMC)-specific miR-17∼92 and PDZ and LIM domain 5 (PDLIM5) knockout mice and overexpressed miR-17∼92 and PDLIM5 by injection of miR-17∼92 mimics or PDLIM5-V5-His plasmids and measured their responses to hypoxia. We used miR-17∼92 mimics, inhibitors, overexpression vectors, small interfering RNAs against PDLIM5, Smad, and transforming growth factor (TGF)-ß to determine the role of miR-17∼92 and its downstream targets in PASMC proliferation and differentiation. MEASUREMENTS AND MAIN RESULTS: We found that human PASMC (HPASMC) from patients with PAH expressed decreased levels of the miR-17∼92 cluster, TGF-ß, and SMC markers. Overexpression of miR-17∼92 increased and restored the expression of TGF-ß3, Smad3, and SMC markers in HPASMC of normal subjects and patients with idiopathic PAH, respectively. Knockdown of Smad3 but not Smad2 prevented miR-17∼92-induced expression of SMC markers. SMC-specific knockout of miR-17∼92 attenuated hypoxia-induced pulmonary hypertension (PH) in mice, whereas reconstitution of miR-17∼92 restored hypoxia-induced PH in these mice. We also found that PDLIM5 is a direct target of miR-17/20a, and hypertensive HPASMC and mouse PASMC expressed elevated PDLIM5 levels. Suppression of PDLIM5 increased expression of SMC markers and enhanced TGF-ß/Smad2/3 activity in vitro and enhanced hypoxia-induced PH in vivo, whereas overexpression of PDLIM5 attenuated hypoxia-induced PH. CONCLUSIONS: We provided the first evidence that miR-17∼92 inhibits PDLIM5 to induce the TGF-ß3/SMAD3 pathway, contributing to the pathogenesis of PAH.

Role of 20-HETE in the impaired myogenic and TGF responses of the Af-Art of Dahl salt-sensitive rats.

The present study examined whether 20-HETE production is reduced in the renal vasculature and whether this impairs myogenic or tubuloglomerular feedback (TGF) responses of the afferent arteriole (Af-Art). The production of 20-HETE was 73% lower in renal microvessels of Dahl salt-sensitive rats (SS) rats than in SS.5(BN) rats, in which chromosome 5 from the Brown Norway (BN) rat containing the CYP4A genes was transferred into the SS genetic background. The luminal diameter of the Af-Art decreased by 14.7 ± 1.5% in SS.5(BN) rats when the perfusion pressure was increased from 60 to 120 mmHg, but it remained unaltered in SS rats. Administration of an adenosine type 1 receptor agonist (CCPA, 1 µM) reduced the diameter of the Af-Art in the SS.5(BN) rats by 44 ± 2%, whereas the diameter of the Af-Art of SS rats was unaltered. Autoregulation of renal blood flow (RBF) and glomerular capillary pressure (PGC) was significantly impaired in SS rats but was intact in SS.5(BN) rats. Administration of a 20-HETE synthesis inhibitor, HET0016 (1 µM), completely blocked the myogenic and adenosine responses in the Af-Art and autoregulation of RBF and PGC in SS.5(BN) rats, but it had no effect in SS rats. These data indicate that a deficiency in the formation of 20-HETE in renal microvessels impairs the reactivity of the Af-Art of SS rats and likely contributes to the development of hypertension induced renal injury.

Common fibroid-associated genes are differentially expressed in phenotypically dissimilar cell populations isolated from within human fibroids and myometrium.

Uterine fibroids are a prevalent gynaecological condition in reproductive-aged women and are the commonest reason for hysterectomy. The cellular composition of clonal fibroids are heterogeneous, with phenotypically dissimilar cells that include smooth muscle cells (SMC), vascular SMC (VSMC) and fibroblasts. The aim of our study was to investigate genes that are commonly differentially expressed between fibroid and myometrial whole tissues in phenotypically different sub-populations of cells isolated from fibroid and myometrium. Genes to be investigated by fluorescence-activated cell sorting, quantitative real-time PCR and immunocytochemistry include transforming growth factor ß (TGFB) and retinoic acid (RA) signalling families and steroid hormone receptors. We hypothesised that each cell population isolated from fibroid and myometrium would differ in the expression of fibroid-associated genes. We demonstrated that phenotypically different cellular constituents of uterine fibroids differentially express cellular RA-binding protein 2 (CRABP2), progesterone receptor B (PRB) and TGFB receptor 2 mRNA in fibroid-derived cells of VSMC and SMC phenotype. CRABP2 mRNA was also differentially expressed in fibroblasts and VSMC sub-populations from within clonal fibroid tumours. We conclude that differential regulation of RA, TGFB and PR pathway transcription occurs in fibroid-associated SMC and -fibroblasts and that investigation of paracrine interactions between different cell types within the fibroid microenvironment provides an important new paradigm for understanding the pathophysiology of this common disease.

Multifunctional bone morphogenetic protein system in endocrinology.

New biological activities of bone morphogenetic proteins (BMPs) in the endocrine system have recently been revealed. The BMP system is composed of approximately 30 ligands and preferential combinations of type I and type II receptors. The BMP system not only induces bone formation but also plays unique tissue-specific roles in various organs. For instance, the ovarian BMP system is a physiological inhibitor of luteinization in growing ovarian follicles. In the ovary, the expression of oocyte-derived BMP-15 is critical for female reproduction. In the pituitary, BMP-4 is a key player for initial development of the anterior pituitary, while it is also functionally involved in some differentiated pituitary tumors, including prolactinoma and Cushing's disease. In the adrenal glands, BMP-6 and BMP-4 modulate aldosterone and catecholamine production, respectively, which contributes to a functional interaction between the cortex and medulla. In the present review, recent advances in BMP biology in the field of endocrinology are described and the possibility for clinical application of BMP activity is discussed.

Role of connexin40 in the autoregulatory response of the afferent arteriole.

Connexins in renal arterioles affect autoregulation of arteriolar tonus and renal blood flow and are believed to be involved in the transmission of the tubuloglomerular feedback (TGF) response across the cells of the juxtaglomerular apparatus. Connexin40 (Cx40) also plays a significant role in the regulation of renin secretion. We investigated the effect of deleting the Cx40 gene on autoregulation of afferent arteriolar diameter in response to acute changes in renal perfusion pressure. The experiments were performed using the isolated blood perfused juxtamedullary nephron preparation in kidneys obtained from wild-type or Cx40 knockout mice. Renal perfusion pressure was increased in steps from 75 to 155 mmHg, and the response in afferent arteriolar diameter was measured. Hereafter, a papillectomy was performed to inhibit TGF, and the pressure steps were repeated. Conduction of intercellular Ca(2+) changes in response to local electrical stimulation was examined in isolated interlobular arteries and afferent arterioles from wild-type or Cx40 knockout mice. Cx40 knockout mice had an impaired autoregulatory response to acute changes in renal perfusion pressure compared with wild-type mice. Inhibition of TGF by papillectomy significantly reduced autoregulation of afferent arteriolar diameter in wild-type mice. In Cx40 knockout mice, papillectomy did not affect the autoregulatory response, indicating that these mice have no functional TGF. Also, Cx40 knockout mice showed no conduction of intercellular Ca(2+) changes in response to local electrical stimulation of interlobular arteries, whereas the Ca(2+) response to norepinephrine was unaffected. These results suggest that Cx40 plays a significant role in the renal autoregulatory response of preglomerular resistance vessels.

Human immunodeficiency virus enhances hepatitis C virus replication by differential regulation of IFN and TGF family genes.

HIV co-infection significantly impacts the natural history of hepatitis C virus (HCV) by increasing plasma HCV viral load, accelerating liver disease progression, and reducing rates of HCV clearance. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the impact of HIV on cytokine expression is unknown. In this study, an HCV continuous infection cell culture system was modified to permit co-infection with HIV to test the hypothesis that virus-induced disregulation of immune-response genes, particularly interferons and TGF-ß, may create a permissive environment for the initial establishment of HIV/HCV co-infection in the host. CCR5-expressing Huh-7.5 hepatoma cells were transduced with human CD4 antigen to allow HIV infection in vitro. Co-infection of CD4⁺ Huh-7.5 cells with HIV and HCV or co-culture of HIV-infected CD4⁺ Huh-7.5 cells and HCV-infected Huh-7.5 cells increased the level of HCV RNA compared to HCV mono-infection. Quantitative gene expression analysis revealed HIV-induced up regulation of most tested IFN family genes when compared to HCV or co-infection. HCV infection induced up regulation of many TGF family genes that were subsequently down-regulated in the presence of HIV or HIV/HCV. Interestingly, co-infection resulted in down regulation of several IFN genes and significant up regulation of TGF-ß genes leading to an overall enhancement of HCV replication. These data suggest that HIV infection may influence HCV replication in vitro by increasing levels of HCV RNA, possibly through the differential regulation of endogenous IFN and TGF family genes.

Cutaneous scarring: Pathophysiology, molecular mechanisms, and scar reduction therapeutics Part I. The molecular basis of scar formation.

Cutaneous scarring is often the epicenter of patient-related concerns, and the question "Will there be a scar?" is one that is all too familiar to the everyday clinician. In approaching this topic, we have reviewed the pathology, the embryology, and the molecular biology of cutaneous scarring.

The physiological role of endoglin in the cardiovascular system.

Endoglin (CD105) is an integral membrane glycoprotein that serves as a coreceptor for members of the transforming growth factor-ß superfamily of proteins. A major role for endoglin in regulating transforming growth factor-ß-dependent vascular remodeling and angiogenesis has been postulated based on the following: 1) endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1, a disease characterized by vascular malformations; 2) endoglin knockout mice die at midgestation because of defective angiogenesis; 3) endoglin is overexpressed in neoangiogenic vessels, during inflammation, and in solid tumors; and 4) endoglin regulates the expression and activity of endothelial nitric oxide synthase, which is involved in angiogenesis and vascular tone. Besides the predominant form of the endoglin receptor (long endoglin isoform), two additional forms of endoglin have been recently reported to play a role in the vascular pathology and homeostasis: the alternatively spliced short endoglin isoform and a soluble endoglin form that is proteolytically cleaved from membrane-bound endoglin. The purpose of this review is to underline the role that the different forms of endoglin play in regulating angiogenesis, vascular remodeling, and vascular tone, as well as to analyze the molecular and cellular mechanisms supporting these effects.

Immune factors in human embryo culture and their significance.

There is increasing evidence that human development before implantation is regulated by embryonically and maternally derived growth factors. The "regulators" of embryonic origin such as soluble human leukocyte antigen G, platelet-activating factor, Th1/Th2 cytokines, insulin-like growth factor, epidermal growth factor, transforming growth factor alpha, colony-stimulating factor, platelet-derived growth factor may be used as indicators of embryo viability and implantation potential. The data prove the influence of growth factors on the development and growth of preimplantation embryos. Though there is a lot of research in the field of biomarkers during folliculogenesis and maternal-fetal interface, only few of them deal with regulators derived from embryonic cells to the cultivation medium. The aim of our study was to summarize the research dealing with immune markers produced by embryos in vitro and to estimate their impact on the cell growth, viability and implantation potential.

Canonical signaling by TGF family members in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.

Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche. Prior studies showed that signaling from members of the transforming growth factor (TGF) superfamily in mesenchymal stromal cells is required for normal niche development. Here, we assessed the impact of TGF family signaling on niche maintenance and stress responses by deleting Smad4 in mesenchymal stromal cells at birth, thereby abrogating canonical TGF signaling. No alteration in the number or spatial organization of CXCL12-abundant reticular (CAR) cells, osteoblasts, or adipocytes was observed in Osx-Cre, Smad4fl/fl mice, and expression of key niche factors was normal. Basal hematopoiesis and stress erythropoiesis responses to acute hemolytic anemia were normal. TGF-ß potently inhibits stromal CXCL12 expression in vitro; however, G-CSF induced decreases in bone marrow CXCL12 expression and subsequent hematopoietic stem/progenitor cell mobilization were normal in Osx-Cre, Tgfbr2fl/fl mice, in which all TGF-ß signaling in mesenchymal stromal is lost. Finally, although a prior study showed that TGF-ß enhances recovery from myeloablative therapy, hematopoietic recovery following single or multiple doses of 5-flurauracil were normal in Osx-Cre, Tgfbr2fl/fl mice. Collectively, these data suggest that TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.

Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture.

When a confluent monolayer of bovine aortic endothelial (BAE) cells is wounded with a razor blade, endothelial cells (ECs) spontaneously move into the denuded area. If bovine pericytes or smooth muscle cells (SMCs) are plated into the denuded area at low density, they block the movement of the ECs. This effect is dependent upon the number of cells plated into the wound area and contact between ECs and the plated cells. Antibodies to transforming growth factor-beta 1 (TGF-beta 1) abrogate the inhibition of BAE cell movement by pericytes or SMCs. TGF-beta 1, if added to wounded BAE cell monolayers, also inhibits cell movement. When cultured separately, BAE cells, pericytes, and SMCs each produce an inactive TGF-beta 1-like molecule which is activated in BAE cell-pericyte or BAE cell-SMC co-cultures. The activation appears to be mediated by plasmin as the inhibitory effect on cell movement in co-cultures of BAE cells and pericytes is blocked by the inclusion of inhibitors of plasmin in the culture medium.

Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms.

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and fibroblasts and may stimulate these cells to express endogenous growth factors, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.

Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells.

Transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2 were found to be potent mitogens for purified rat Schwann cells, each stimulating DNA synthesis in quiescent cells and also increasing their proliferation rate. Half-maximal stimulation of DNA synthesis occurred at approximately 0.1 ng/ml TGF-beta 1 or TGF-beta 2. Mitogenic stimulation by TGF-beta 1 and TGF-beta 2 was enhanced by forskolin, which activates adenylate cyclase, at concentrations up to 0.5 microM forskolin. However, at 5 microM forskolin, the synergistic interaction between forskolin and TGF-beta 1 was abolished. These results are in contrast to the observed synergy between forskolin and another Schwann cell mitogen, glial growth factor (GGF). Both 0.5 and 5 microM forskolin were found to enhance the stimulation of DNA synthesis by partially purified GGF (GGF-CM). As well as being functionally distinct, TGF-beta 1 and GGF-CM activities were also physically separable by chromatography on a Superose 12 gel permeation column. Thus, TGF-beta 1 and beta 2 are rat Schwann cell mitogens, and Schwann cells are one of the few normal cell populations to respond mitogenically to TGF-beta.

Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-beta gene families.

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.

Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur.

We have investigated the ability of exogenous transforming growth factor-beta (TGF-beta) to induce osteogenesis and chondrogenesis, critical events in both bone formation and fracture healing. Daily injections of TGF-beta 1 or 2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis. After cessation of the injections, endochondral ossification occurred, resulting in replacement of cartilage with bone. Gene expression of type II collagen and immunolocalization of types I and II collagen were detected within the TGF-beta-induced cartilage and bone. Moreover, injection of TGF-beta 2 stimulated synthesis of TGF-beta 1 in chondrocytes and osteoblasts within the newly induced bone and cartilage, suggesting positive autoregulation of TGF-beta. TGF-beta 2 was more active in vivo than TGF-beta 1, stimulating formation of a mass that was on the average 375% larger at a comparable dose (p less than 0.001). With either TGF-beta isoform, the dose of the growth factor determined which type of tissue formed, so that the ratio of cartilage formation to intramembranous bone formation decreased as the dose was lowered. For TGF-beta 1, reducing the daily dose from 200 to 20 ng decreased the cartilage/intramembranous bone formation ratio from 3.57 to zero (p less than 0.001). With TGF-beta 2, the same dose change decreased the ratio from 3.71 to 0.28 (p less than 0.001). These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGF-beta to proliferate and differentiate, as occurs in embryologic bone formation and early fracture healing.

Structure and function of human amphiregulin: a member of the epidermal growth factor family.

The complete amino acid sequence of amphiregulin, a bifunctional cell growth modulator, was determined. The truncated form contains 78 amino acids, whereas a larger form of amphiregulin contains six additional amino acids at the amino-terminal end. The amino-terminal half of amphiregulin is extremely hydrophilic and contains unusually high numbers of lysine, arginine, and asparagine residues. The carboxyl-terminal half of amphiregulin (residues 46 to 84) exhibits striking homology to the epidermal growth factor (EGF) family of proteins. Amphiregulin binds to the EGF receptor but not as well as EGF does. Amphiregulin fully supplants the requirement for EGF or transforming growth factor-alpha in murine keratinocyte growth, but it is a much weaker growth stimulator in other cell systems.

Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis.

Directed cell migration is important for many aspects of normal animal development, but little is known about how cell migrations are guided or the mechanisms by which guidance cues are translated into directed cell movement. Here we present evidence that signaling mediated by the epidermal growth factor receptor (EGFR) guides dorsal migration of border cells during Drosophila oogenesis. The transforming growth factor-alpha (TGF-alpha)-like ligand Gurken appears to serve as the guidance cue. To mediate this guidance function, EGFR signals via a pathway that is independent of Raf-MAP kinase and receptor-specific.

Stromal reprogramming: A target for tumor therapy.

Cancer associated fibroblasts (CAFs) as the dominant, long-lived and highly plastic cells within the tumor microenvironment (TME) with multi-faceted roles that are endowed with tumor aggressive features. They can instruct and shape the stroma of tumor into being a highly qualified bed for cellular recruitment, differentiation and plasticity in the host tissue or secondary organ/s. In this Review, we have a discussion over CAF reprogramming as a general concept, inducers and outcomes, pursued by suggesting potential strategies to combat this key promoter of tumor.

Growth stimulation by coexpression of transforming growth factor-alpha and epidermal growth factor-receptor in normal and adenomatous human colon epithelium.

Autocrine stimulation of the epidermal growth factor receptor (EGF-R), by coexpression of transforming growth factor-alpha (TGF-alpha), causes malignant transformation of some fibroblast cell lines. TGF-alpha and EGF-R are both known to be expressed in colon carcinoma tissue and have been shown coexpressed in colon carcinoma cell lines. TGF-alpha autocrine activation of EGF-R has been suggested as a potential mechanism contributing to abnormal growth control in colon cancer. We now report coexpression of TGF-alpha and EGF-R transcripts in morphologically normal colonic epithelium from five individuals, in colonic adenomas from three individuals, and in a nontumorigenic colon adenoma cell line, VACO-330. Functional studies demonstrate VACO-330 growth is stimulated by exogenous TGF-alpha and is completely abolished by a blocking anti-EGF-R antibody. Autocrine stimulation of EGF-R by TGF-alpha is therefore required for growth of the adenoma cell line. Autocrine stimulation of EGF-R by TGF-alpha does not cause malignant transformation of the colonic epithelial cell. In normal and adenomatous human colon TGF-alpha, via either an autocrine or paracrine mechanism, is likely an important physiologic stimulant of epithelial proliferation.

Differential expression of transforming growth factors alpha and beta in rat intestinal epithelial cells.

Expression of transforming growth factor alpha (TGF alpha), and transforming growth factor beta (TGF beta) was assessed in isolated primary rat intestinal epithelial cells as well as a rat intestinal crypt cell-derived cell line (IEC-6). A gradient in TGF beta was present, with high concentrations of a 2.5-kb transcript found in undifferentiated crypt cells and progressively lower amounts of the TGF beta transcript in increasingly differentiated villus cell populations. In contrast, the concentration of 4.5-kb TGF alpha transcript was higher in differentiated villus cells than in mitotically active, undifferentiated populations of crypt epithelial cells. The concentrations of transforming growth factors alpha and beta as determined by radioreceptor binding inhibition assay and direct assessment of transforming growth factor biological activity correlated with Northern blot analysis. Although gradients in the expression of the TGFs were present, equivalent binding was observed in the different intestinal cell populations when assessed with 125I-TGF beta and 125I-EGF (TGF alpha). No EGF transcripts were detected in any intestinal cell population, suggesting that the true ligand of the EGF receptor was TGF alpha. IEC-6 cells expressed both TGF alpha and TGF beta transcripts. In addition to the transcripts identified in the primary intestinal cells, this cell line contained an additional larger TGF alpha transcript (4.8 kb) and smaller TGF beta transcripts (2.2 and 1.8 kb). TGF alpha and TGF beta may play a significant role in the regulation of the balance between proliferative and differentiated cell compartments in the intestinal epithelium through both autocrine and paracrine mechanisms.