Arnold E. Postlethwaite

Transforming growth factor-beta increases steady state levels of type I procollagen and fibronectin messenger RNAs posttranscriptionally in cultured human dermal fibroblasts.

Transforming growth factor-beta (TGF beta), when injected subcutaneously into newborn mice, induces a rapid fibrotic response, stimulates chemotaxis, and elevates the rates of biosynthesis of collagen and fibronectin by fibroblasts in vitro. We explored the molecular mechanisms of TGF beta-mediated stimulation of collagen and fibronectin synthesis in cultured human foreskin fibroblasts. TGF beta preferentially stimulated the synthesis of fibronectin and type I procollagen chains 3-5-fold as shown by polypeptide analysis. Concomitant elevation in the steady state levels of messenger RNAs (mRNAs) coding for type I procollagen and fibronectin also occurred but without a net increase in the rate of transcription of either of these genes. The preferential stabilization of mRNAs specifying type I procollagen and fibronectin provides a partial explanation for the mechanisms by which TGF beta enhances the synthesis of type I procollagen and fibronectin in mesenchymal cells.

Infarct scar as living tissue.

Abstract. Infarct scar tissue has long been considered inert (acellular, composed simply of fibrillar collagen) and whose function is simply to restore structural integrity to infarcted myocardium and to provide tensile strength that prevents tissue rupture. Technologies of cellular and molecular biology have altered this perspective. Infarct scar is now recognized as living tissue: composed of a persistent population of fibroblast-like cells whose ongoing activity includes a regulation of collagen turnover and scar tissue contraction and which are nourished by a neovasculature. Herein we briefly review these various components of the infarct scar that provide for its dynamic nature and which is relevant to todays interest in preventing heart failure through a rebuilding (regrowing) of myocardial tissue at the infarct size.

Fibroblast chemotaxis induction by human recombinant interleukin-4. Identification by synthetic peptide analysis of two chemotactic domains residing in amino acid sequences 70-88 and 89-122.

Interleukin-4 is a T lymphocyte- and mast cell-derived cytokine with pleiotropic properties with biological effects on a variety of target cells including B and T lymphocytes, macrophages, hematopoietic cells, mast cells, and fibroblasts. In addition to the proliferation effect of IL-4 on fibroblasts, which has been previously described, in this report the chemotactic properties of IL-4 for fibroblasts is described. Human recombinant IL-4 induced the chemotactic migration of dermal fibroblasts in vitro in modified Boyden-type chambers at concentrations between 10(-12) and 10(-11) M. The chemotactic activity of IL-4 was neutralized by anti-human recombinant IL-4 IgG antibodies. Oligopeptides representing the complete deduced amino acid sequence of human IL-4 were synthesized by the Merrifield technique and tested for their ability to induce fibroblast chemotaxis. Two peptides representing residues 70-88 and 89-122 induced fibroblast migration. Peptide 70-88 was the more potent of the two causing chemotaxis of fibroblasts at 10(-8)-10(-6) M while peptide 89-129 induced migration at 10(-7)-10(-5) M. Although the mechanism by which IL-4 and these two peptides induce fibroblast chemotaxis is unknown, each of these three compounds were able to chemotactically desensitize fibroblasts to the chemotactic effects of the other two but not to a structurally unrelated chemotactic cytokine, transforming growth factor beta-1. These studies suggest that IL-4 might function in vivo to induce the accumulation of fibroblasts at sites of tissue injury, inflammatory and immune reactions in which T lymphocytes and mast cells participate.

Cellular origins of fibroblasts : Possible implications for organ fibrosis in systemic sclerosis

Purpose of reviewThere is an intense interest in the potential of circulating blood cells and epithelium-related nonfibroblast cells to change into matrix synthesizing fibroblasts and myofibroblasts. These sources of fibroblasts may have importance in systemic sclerosis (scleroderma). Recent findingsEpithelial cells from different sources can transition into fibroblasts and myofibroblasts in response to transforming growth factor β and other growth factors/cytokines. This is called epithelial-mesenchymal transition (EMT). EMT has been repeatedly demonstrated to occur in several models of renal fibrosis including lupus prone mice. Quite unexpectedly, bone morphogenic protein 7 prevents EMT and protects lupus mice and other renal fibrosis models from developing fibrosis in the kidneys. Human peripheral blood mononuclear cells under different conditions of culture give rise to several different types of fibroblast-like cells. In SSc, it has been observed that the sera have low levels of serum amyloid protein. Serum amyloid protein has been found to inhibit the generation of fibrocytes from CD14+ precursors. The implications of these potential sources of fibroblasts and myofibroblasts in systemic sclerosis and related rheumatic diseases are discussed. SummaryFibroblasts and myofibroblasts in skin and internal organs of patients with systemic sclerosis and related diseases may possibly arise not only from the resident fibroblast population but from epithelial cells, pericytes, monocytes, and other progenitors from the circulating pool of hematopoietic cells and stem cells. These alternative sources of fibroblasts would best be treated by specifically targeting the transition or transdifferentiation process by which cells change into fibroblasts.

In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1

We define previously unrecognized in vivo pathways of vitamin D3 (D3) metabolism generating novel D3‐hydroxyderivatives different from 25‐hydroxyvitamin D3 [25(OH)D3] and 1,25(OH)2D3. Their novel products include 20‐hydroxyvitamin D3 [20(OH)D3], 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, 1,20(OH)2D3,1,20,23(OH)3D3, and 17,20,23(OH)3D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ~20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α‐hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)2D3 → 17,20,23(OH)3D3 with further 1α‐hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)2D3 and D3 → 22(OH)D3 → 20,22(OH)2D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)2D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)2D3.—Slominski, A. T., Km, T.‐K., Shehabi, H. Z., Semak, I., Tang, E. K. Y., Nguyen, M. N., Benson, H. A. E., Korik, E., Janjetovic, Z., Chen, J., Yates, C. R., Postlethwaite, A., Li, W., Tuckey, R. C. In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1. FASEB J. 26, 3901–3915 (2012). www.fasebj.org

Aldosteronism and Peripheral Blood Mononuclear Cell Activation: A Neuroendocrine-Immune Interface

Abstract— Aldosteronism eventuates in a proinflammatory/fibrogenic vascular phenotype of the heart and systemic organs. It remains uncertain whether peripheral blood mononuclear cells (PBMCs) are activated before tissue invasion by monocytes/macrophages and lymphocytes, as is the case for responsible pathogenic mechanisms. Uninephrectomized rats treated for 4 weeks with dietary 1% NaCl and aldosterone (ALDOST, 0.75 &mgr;g/h) with or without spironolactone (Spi, 100 mg/kg per daily gavage) were compared with unoperated/untreated and uninephrectomized/salt-treated controls. Before intramural coronary vascular lesions appeared at week 4 of ALDOST, we found (1) a reduction of PBMC cytosolic free [Mg2+]i, together with intracellular Mg2+ and Ca2+ loading, whereas plasma and cardiac tissue Mg2+ were no different from controls; (2) increased H2O2 production by monocytes and lymphocytes together with upregulated PBMC gene expression of oxidative stress–inducible tyrosine phosphatase and Mn2+-superoxide dismutase and the presence of 3-nitrotyrosine in CD4+ and ED-1–positive inflammatory cells that had invaded intramural coronary arteries; (3) B-cell activation, including transcription of immunoglobulins, intracellular adhesion molecule-1, and CC and CXC chemokines and their receptors; (4) expansion of B lymphocyte subset and myosin heavy chain class II–expressing lymphocytes; and (5) autoreactivity with gene expression for antibodies to acetylcholine receptors and a downregulation of RT-6.2, which is in keeping with cell activation and associated with autoimmunity. Spi cotreatment attenuated the rise in intracellular Ca2+, the appearance of oxidative/nitrosative stress in PBMCs and invading inflammatory cells, and alterations in PBMC transcriptome. Thus, aldosteronism is associated with an activation of circulating immune cells induced by iterations in PBMC divalent cations and transduced by oxidative/nitrosative stress. ALDO receptor antagonism modulates this neuroendocrine-immune interface. The full text of this article is available online at http://www.circresaha.org.

Cell-mediated immunity to collagen and collagen α chains in rheumatoid arthritis and other rheumatic diseases

Peripheral blood mononuclear cells from patients with rheumatoid arthritis, gout, ankylosing spondylitis and degenerative joint disease were cultured in the presence of native types I, II and III collagens and alpha chains from each of these types of collagen. The culture supernatant fluids were harvested and assayed for lymphocyte-derived chemotatic factor for monocytes. Reactions to one or more of the native collagens was found in 50 per cent (10 of 20) of the patients with rheumatoid arthritis, 20 per cent (two of 10) of the patients with gout and ankylosing spondylitis but in none of the 10 patients with degenerative joint disease or in normal subjects. Reaction to one or more alpha chains was found in 90 per cent (18 of 20) of the patients with rheumatoid arthritis, 60 per cent (six of 10) of the patients with gout, 50 per cent (five of 10) of the patients with ankylosing spondylitis, 30 per cent (three of 10) of the patients with degenerative joint disease and in 10 per cent of the normal subjects (one of 10). All the reactions were quantiatively stronger in patients with rheumatoid arthritis. These results indicate that patients with rheumatoid arthritis have cell-mediated immunity to homologous native and denatured collagens but that the reaction is not specific for rheumatoid arthritis. Some patients with gout, ankylosing spondylitis and degenerative joint disease also have low levels of immunity.

Pathogenesis of systemic sclerosis

Systemic scleroderma (SSc) is one of the most complex systemic autoimmune diseases. It targets the vasculature, connective tissue-producing cells (namely fibroblasts/myofibroblasts), and components of the innate and adaptive immune systems. Clinical and pathologic manifestations of SSc are the result of: (1) innate/adaptive immune system abnormalities leading to production of autoantibodies and cell-mediated autoimmunity, (2) microvascular endothelial cell/small vessel fibroproliferative vasculopathy, and (3) fibroblast dysfunction generating excessive accumulation of collagen and other matrix components in skin and internal organs. All three of these processes interact and affect each other. The disease is heterogeneous in its clinical presentation that likely reflects different genetic or triggering factor (i.e., infection or environmental toxin) influences on the immune system, vasculature, and connective tissue cells. The roles played by other ubiquitous molecular entities (such as lysophospholipids, endocannabinoids, and their diverse receptors and vitamin D) in influencing the immune system, vasculature, and connective tissue cells are just beginning to be realized and studied and may provide insights into new therapeutic approaches to treat SSc.

LACK OF EFFICACY OF ORAL BOVINE TYPE II COLLAGEN ADDED TO EXISTING THERAPY IN RHEUMATOID ARTHRITIS

OBJECTIVE To investigate the efficacy of oral type II collagen (CII) in the treatment of rheumatoid arthritis (RA), when added to existing therapy. METHODS Patients with active RA (n = 190) were randomized into a 6-month, double-blind, placebo-controlled trial. Patients continued to take their current arthritis medications. Patients received either placebo or bovine CII, 0.1 mg/day for 1 month, then 0.5 mg/day for 5 months. RESULTS There were no significant differences between the baseline characteristics of either group. The primary response parameter was the American College of Rheumatology (ACR) preliminary definition of improvement in RA (ACR 20). There was no statistically significant difference in the ACR 20 after 6 months (20.0% of placebo patients; 16.84% of bovine CII patients). There were significant differences in several clinical variables after treatment, all favoring the placebo group. CONCLUSION Oral solubilized bovine CII, added to existing therapy, did not improve disease activity in patients with RA.

RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20‐hydroxyvitamin D3 [20(OH)D3] and 20,23‐dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet‐on RORα or RORγ expression vector and a ROR‐responsive element (RORE)‐LUC reporter, and a mammalian 2‐hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD‐interacting LXXLL‐peptide, were used to study ROR‐antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20, 23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE‐mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL‐17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.—Slominski, A. T., Kim, T.‐K., Takeda, Y., Janjetovic, Z., Brożyna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20‐hydroxy‐ and 20,23‐dihydroxyvitamin D. FASEB J. 28, 2775–2789 (2014). www.fasebj.org