Matthew R. Duncan

Connective tissue growth factor mediates transforming growth factor β-induced collagen synthesis: down-regulation by cAMP

Connective tissue growth factor (CTGF) is a cysteine‐rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF‐β) that acts as a downstream mediator of TGF‐β‐induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF‐β‐induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF‐β stimulated collagen synthesis. Moreover, TGF‐β‐induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti‐CTGF antibodies and by suppressing TGF‐β‐induced CTGF gene expression by elevating intracellular cAMP levels with either membrane‐permeable 8‐Br‐cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF‐induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF‐β activation of a human CTGF promoter/lacZ reporter transgene. Both 8‐Br‐cAMP and CTX blocked TGF‐β‐induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF‐β in neonatal mice, but not increases induced by CTGF or TGF‐β combined with CTGF. Our data indicate that CTGF mediates TGF‐β‐induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF‐β‐induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation.—Duncan, M. R., Frazier, K. S. Abramson, S., Williams, S., Klapper, H., Huang, X., Grotendorst, G. R. Connective tissue growth factor mediates transforming growth factor β‐induced collagen synthesis: down‐regulation by cAMP. FASEB J. 13, 1774–1786 (1999)

Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation

Fibroblast proliferation, differentiation into myofibroblasts, and increased collagen synthesis are key events during both normal wound repair and fibrotic lesion formation. Here we report that these biological responses to TGF–β by fibroblasts are regu¬lated via a CTGF–dependent pathway in concert with either EGF or IGF–2. Our studies indicate these re¬sponses to TGF–β are mutually exclusive, and cells that are proliferating do not express α–SMA or elevated levels of collagen synthesis. Cells expressing α–SMA do not exhibit DNA synthesis but do coexpress higher levels of types I and III collagen mRNA Thus, fibro–blast proliferation and differentiation are controlled by combinatorial signaling pathways involving not only components of the TGF–β/CTGF pathway, but also signaling events induced by EGF and IGF–2–activated receptors. Collectively, our studies indicate TGF–β functions as a classic embryonic inducer, initiating a cascade that is controlled by other factors in the cellular environment. We propose a model for this process with regard to wound repair and fibrotic lesion formation that is likely applicable to other instances of CTGF action during embryogenesis.—Grotendorst, G. R., Rahmanie, H., Duncan, M. R. Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation.

Connective tissue growth factor acts within both endothelial cells and β cells to promote proliferation of developing β cells

Type 1 and type 2 diabetes result from an absolute or relative reduction in functional β-cell mass. One approach to replacing lost β-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance β-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic β cells that we previously showed is required for β-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or β cells results in decreased embryonic β-cell proliferation, making CTGF unique as an identified β cell-derived factor that regulates embryonic β-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in β-cell proliferation. Furthermore, CTGF overexpression in β cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature β cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature β cells in directed differentiation or regeneration protocols.

Recombinant CCN1 prevents hyperoxia-induced lung injury in neonatal rats

BackgroundCystein-rich protein 61 (Cyr61/CCN1) is a member of the CCN family of matricellular proteins that has an important role in tissue development and remodeling. However, the role of CCN1 in the pathogenesis of bronchopulmonary dysplasia (BPD) is unknown. Accordingly, we have investigated the effects of CCN1 on a hyperoxia-induced lung injury model in neonatal rats.MethodsIn experiment 1, newborn rats were randomized to room air (RA) or 85% oxygen (O2) for 7 or 14 days, and we assessed the expression of CCN1. In experiment 2, rat pups were exposed to RA or O2 and received placebo or recombinant CCN1 by daily intraperitoneal injection for 10 days. The effects of CCN1 on hyperoxia-induced lung inflammation, alveolar and vascular development, vascular remodeling, and right ventricular hypertrophy (RVH) were observed.ResultsIn experiment 1, hyperoxia downregulated CCN1 expression. In experiment 2, treatment with recombinant CCN1 significantly decreased macrophage and neutrophil infiltration, reduced inflammasome activation, increased alveolar and vascular development, and reduced vascular remodeling and RVH in the hyperoxic animals.ConclusionThese results demonstrate that hyperoxia-induced lung injury is associated with downregulated basal CCN1 expression, and treatment with CCN1 can largely reverse hyperoxic injury.

Riociguat prevents hyperoxia-induced lung injury and pulmonary hypertension in neonatal rats without effects on long bone growth

Bronchopulmonary dysplasia (BPD) remains the most common and serious chronic lung disease of premature infants. Severe BPD complicated with pulmonary hypertension (PH) increases the mortality of these infants. Riociguat is an allosteric soluble guanylate cyclase stimulator and is approved by the FDA for treating PH in adults. However, it has not been approved for use in neonates due to concern for adverse effects on long bone growth. To address this concern we investigated if administration of riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without side effects on long bone growth in newborn rats. Newborn rats were randomized to normoxia (21% O2) or hyperoxia (85% O2) exposure groups within 24 hours of birth, and received riociguat or placebo by once daily intraperitoneal injections during continuous normoxia or hyperoxia exposure for 9 days. In the hyperoxia control group, radial alveolar count, mean linear intercept and vascular density were significantly decreased, the pathological hallmarks of BPD, and these were accompanied by an increased inflammatory response. There was also significantly elevated vascular muscularization of peripheral pulmonary vessels, right ventricular systolic pressure and right ventricular hypertrophy indicating PH. However, administration of riociguat significantly decreased lung inflammation, improved alveolar and vascular development, and decreased PH during hyperoxia by inducing cGMP production. Additionally, riociguat did not affect long bone growth or structure. These data indicate that riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without affecting long bone growth and structure and hence, suggests riociguat may be a potential novel agent for preventing BPD and PH in neonates.