Connective tissue growth factor (CTGF) is a 36-to 38-kDa peptide that is selectively induced by transforming growth factor-beta (TGF-beta) in fibroblastic cell types. We compared the biologic activities of CTGF with TGF-beta on fibroblasts in culture and in animal models of fibroplasia. CTGF was active as a mitogen in monolayer cultures of normal rat kidney fibroblasts. CTGF did not stimulate anchorage-independent growth of NRK fibroblasts, however, or inhibit the growth of mink lung epithelial cells, distinguishing CTGF's growth-regulatory activities from those of TGF-beta. In NRK fibroblasts, both TGF-beta and CTGF significantly increased the transcripts encoding alpha 1 type I collagen, alpha 5 integrin, and fibronectin. Stimulation of type I collagen and fibronectin protein synthesis by TGF-beta and CTGF was confirmed by pulse labeling of cells with [35S]methionine. Subcutaneous injection of TGF-beta and CTGF into neonatal NIH Swiss mice resulted in a large stimulation of granulation tissue and fibrosis at the site of injection. In situ hybridization studies revealed that TGF-beta injection induced high levels of CTGF mRNA in the dermal fibroblasts at the injection site, demonstrating that TGF-beta can induce the expression of CTGF in connective tissue cells in vivo. No CTGF transcripts were detected in the epidermal cells in either control or TGF-beta-injected skin or in fibroblasts in control (saline-injected) skin. These results demonstrate that, like TGF-beta, CTGF can induce connective tissue cell proliferation and extracellular matrix synthesis.

Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-beta) that acts as a downstream mediator of TGF-beta-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-beta-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-beta stimulated collagen synthesis. Moreover, TGF-beta-induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti-CTGF antibodies and by suppressing TGF-beta-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-beta activation of a human CTGF promoter/lacZ reporter transgene. Both 8-Br-cAMP and CTX blocked TGF-beta-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF-beta in neonatal mice, but not increases induced by CTGF or TGF-beta combined with CTGF. Our data indicate that CTGF mediates TGF-beta-induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF-beta-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation.

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Abstract. Suppurative, ulcerative endometritis associated with bovine herpesvirus-4 (BHV-4) infection was identified in 15 postparturient dairy cows from 5 separate dairies. Characteristic eosinophilic to amphophilic intranuclear viral inclusion bodies were identified within degenerate endometrial lining epithelium and endothelial cells. Bovine herpesvirus-4 was confirmed as the etiology by a combination of fluorescent antibody assays, viral isolation, heminested PCR, ultrastructural examination of the uterus and inoculated tissue culture cells, and negative-stain electron microscopy of tissue culture supernatant. Viral particles measuring 70-95 nm were demonstrated in uterine epithelial and endothelial cells by electron microscopy. Bacteria including Arcanobacterium pyogenes, Escherichia coli, and an ␣-Streptococcus isolate were isolated from all uteri. Bovine herpesvirus-4-associated endometritis has been previously reported in sporadic cases in Europe but has not been previously reported in the United States. Endometritis associated with BHV-4 appears to be an emerging syndrome in Georgia dairy herds.

Abstract. Connective tissue growth factor (CTGF) has been shown to mediate many actions of transforming growth factor-beta (TGF-␤) in the fibrotic response in several diseases. We compared expression of CTGF, TGF-␤, platelet-derived growth factor (PDGF), TNF-␣, and interleukin-1 (IL-1) by in situ hybridization in Sprague-Dawley rats euthanized at 0, 2, 4, and 8 weeks after 5/6 nephrectomy using the rat remnant kidney model of renal failure. Collagen was evaluated by trichrome stains, immunohistochemistry, and electron microscopy. We compared expression patterns to cells undergoing metaplasia. Tubular epithelial regeneration and transdifferentiation to myofibroblasts were assessed morphologically and by proliferating cell nuclear antigen, smooth muscle actin, desmin, and vimentin immunohistochemistry. CTGF expression was minimal in controls, mild at 2 weeks and marked by 4 to 8 weeks in interstitial fibroblasts, coinciding with damage, regeneration, and fibrosis. TGF-␤ expression was increased in many cell types at 2 weeks, increased further by 4 weeks, then remained constant. PDGF-B messenger RNA was found in many stromal cells at 2-4 weeks, but expression decreased at 8 weeks. No significant IL-1 or TNF-␣ staining was detected. We conclude that CTGF and interacting factors are associated with development or progression of chronic interstitial fibrosis. Proximity of CTGF, TGF-␤, and PDGF mRNA expression to regenerative epithelial cells and those transdifferentiating to myofibroblasts suggests that growth factors may modulate renal tubular epithelial differentiation.