Conclusions: A remarkable observation was the development of extensive fibrosis in the skin, lungs and other organs in mice with constitutive endothelial cell-specific activation of TGF-β-signaling. Alterations resembling the microvascular pathology characteristic of human SSc were also observed. These results render this transgenic mouse strain a valuable model for SSc as they reproduce the typical cutaneous and visceral tissue fibrosis and severe fibroproliferative microvascular alterations found in SSc. These results also provide new information about the pathogenesis of tissue fibrosis in response to the effect of increased TGF-β expression specifically in cells of endothelial origin. (Supported by NIH Grant AR055660 to SAJ.)

Peter J. Wermuth, PhD, Kellan R. Carney, BS, Fabian A. Mendoza, MD, Sonsoles Piera-Velazquez, PhD, and Sergio A. Jimenez, MD. Jefferson Institute of Molecular Medicine and Division of Connective Tissue Diseases and Scleroderma Center, Thomas Jefferson University, Philadelphia, Pa., USA.

Background/Purpose: Microvascular damage is an early event in Systemic Sclerosis (SSc) pathogenesis and may represent the initiating stimulus for the subsequent establishment and progression of the fibrotic process. Extensive experimental evidence shows the increased expression and production of the pleiotropic growth factor transforming growth factor-β (TGF-β) and of TGF-β-regulated genes in SSc patient tissues however, the effects of cell-specific TGF-β expression in endothelial cells have not been studied. The purpose of these studies was to generate a transgenic mouse strain characterized by the inducible expression of TGF-β signaling specifically in cells of endothelial lineage. The novel transgenic mouse strain (TGFβca-Cdh5 Cre) would be utilized for the evaluation of the effect of upregulated TGF-β expression specifically in the microvasculature and to examine directly the role of endothelial cell activation in the development of tissue fibrosis.

Methods: A transgenic mouse strain carrying an inducible constitutively active TGF-β receptor I (TBRIca) allele was intercrossed with a second mouse strain (B6.Cg-Tg(Cdh5-cre/ERT2)Mlia/J) carrying a Cre-ERT2 expression cassette controlled by the endothelial cell-specific Cdh5 promoter. A functional TBRIca allele is generated by intraperitoneal injection of 4-OH tamoxifen, yielding constitutive and unrestricted TGF-β signaling in all cells of endothelial lineage. The results of the intercross were evaluated by histopathologic staining of skin and visceral tissues, by immunhistochemical staining of lung for α-smooth muscle actin and von Willebrand factor, measurement of tissue hydroxyproline content, and by evaluation of the expression of genes associated with tissue fibrosis, myofibroblast differentiation and TGF-β signaling.

Results: Constitutive TGFβ-1 signaling in endothelial lineage cells resulted in severe and progressive cutaneous and visceral tissue fibrosis compared to saline-injected control mice. Increased collagen deposition and microvascular fibroproliferative changes in the dermis, lungs, myocardium, liver, and kidney were observed by histopathological analysis of these tissues. A 2.2 fold increase in hydroxyproline content of the skin and a 2.8 fold increase in the lung was observed. Increased expression of several profibrotic genes associated with tissue fibrosis and the transdifferentiation and activation of myofibroblasts was demonstrated in total RNA isolated from skin and lungs of these animals.

Conclusions: A remarkable observation was the development of extensive fibrosis in the skin, lungs and other organs in mice with constitutive endothelial cell-specific activation of TGF-β-signaling. Alterations resembling the microvascular pathology characteristic of human SSc were also observed. These results render this transgenic mouse strain a valuable model for SSc as they reproduce the typical cutaneous and visceral tissue fibrosis and severe fibroproliferative microvascular alterations found in SSc. These results also provide new information about the pathogenesis of tissue fibrosis in response to the effect of increased TGF-β expression specifically in cells of endothelial origin.

Results Constitutive TGFβ-1 signaling in endothelial

lineage cells resulted in severe and progressive cutaneous and visceral tissue fibrosis compared to saline-injected control mice.

Increased collagen deposition and microvascular fibroproliferative changes in the dermis, lungs, myocardium, liver, and kidney were observed by histopathological analysis of these tissues.

A 2.2 fold increase in hydroxyproline content of the skin and a 2.8 fold increase in the lung was observed.

Increased expression of several profibrotic genes associated with tissue fibrosis and the transdifferentiation and activation of myofibroblasts was demonstrated in the skin and lungs of these animals.

(A) Skin. Sections from control mice and from tamoxifen-injected TBRIca-Cdh5-Cre mice display increased dermal thickness and perivascular collagen accumulation. (B) Lung. Sections from control mice. Sections from TBRIca-Cdh5-Cre tamoxifen-injected mice. Note marked loss of alveolar morphology with tissue consolidation, thickening of alveolar septae, and perivascular and interstitial collagen accumulation.

Sections from heart (A), liver (B), and kidney (C) from control mice and from TBRIca-Cdh5-Cre-tamoxifen-injected mice. The solid box in the center panel indicates the region of magnification for the right panel.

CUTANEOUS AND VISCERAL FIBROSIS INDUCED BY ENDOTHEIAL CELL-SPECIFIC CONSTITUTIVE ACTIVATION OF TGF-β1 SIGNALING IN MICE

Expression of Col1a1, Col3a1, Col4a1, and Fn in skin (A) and lung (B). The values shown are the mean (+/- SD) fold change levels of gene expression from each treatment group performed in triplicate for each tissue. Gene expression levels were normalized to 18S levels. Values for other samples are expressed relative to the control female group which was arbitrarily set at the 100% expression level. Significance was determined by Student’s T-test. Statistical significance: *:p<0.05, **: p<0.01, ***: p<0.001.

Skin sections (A) and lung sections (B) from control mice and from TBRIca-Cdh5-Cre-tamoxifen-injected mice. Note extensive collagen accumulation in the subendothelial space and surrounding vessels as well as marked narrowing with almost complete obliteration of vascular lumen by intensely-staining collagen fibrotic tissue.

Hydoxyproline content of skin (A), lung (B), kidney (C), heart (D) or liver (E) sections isolated from each treatment group of control females (N=4), tamoxifen-injected females (N=7), control males (N=3), or tamoxifen-injected males (N=6) were hydrolyzed and analyzed for hydroxyproline content (µg/mg of tissue wet weight performed in triplicate. Significance determined by Student’s two-tailed t test. *: p<0.05, **: p<0.01, ***: p<0.001.

Expression of Acta2, Fn-Eda, and Comp in skin (A) and lung (B). The values shown are the mean (+/- SD) fold change levels of gene expression from each treatment group performed in triplicate for each tissue. Significance was determined by Student’s T-test. Statistical significance: *:p<0.05, **: p<0.01, ***: p<0.001.

Expression of Snai1, Snai2, Twist, and Mrtfa from skin (A) and lung (B). The values shown are the mean (+/- SD) fold change levels of gene expression from each treatment group performed in triplicate for each tissue. Significance was determined by Student’s T-test. Statistical significance: *:p<0.05, **: p<0.01, ***: p<0.001.

Confocal microscopy staining for vWF (red) and α-SMA (green) in the lungs from TGF-βcaCdh5-Cre control transgenic male mice (A&C), or tamoxifen-injected mice (B&D). DAPI was used for counterstaining of nuclei. Magnification: 40x in A&B; 60X in C&D. Endothelial cells expressing vWF (red) are seen lining the large and small vessels of the lung. Smooth muscle cells expressing α-SMA (green) are seen only in the vessel medial layer in the control samples (A&C) but α-SMA expressing myofibroblasts are present in the endothelial layer of the tamoxifen-injected mice (B&D). Cells co-staining for vWF and α-SMA (yellow; white arrows) are present in the endothelial layer of small vessels of the tamoxifen-treated animals (B&D). No cells co-staining for vWF and α-SMA were found in lungs from control mice (A&C).

Histopathology of skin and lung from control and tamoxifen- injected TBRIca-Cdh5-Cre transgenic mice. Introduction

• Microvascular damage is an early event in Systemic Sclerosis (SSc) pathogenesis and may represent the initiating stimulus for the subsequent establishment and progression of the fibrotic process.

• The effects of cell-specific TGF-β expression in endothelial cells on tissue fibrosis have not been studied.

• The purpose of these studies was to generate a transgenic mouse strain characterized by the inducible expression of TGF-β signaling specifically in cells of endothelial lineage to evaluate the effect of upregulated TGF-β expression specifically in the microvasculature in the development of tissue fibrosis.

Histopathology of kidney, heart and liver from control and tamoxifen-injected TBRIca-Cdh5- Cre transgenic mice.

Histopathology of skin and lung micro- vasculature from control and tamoxifen- injected TBRIca-Cdh5-Cre transgenic mice.

Hydroxyproline content of tissues from control and tamoxifen-injected TBRIca-Cdh5-Cre mice.

Immunohistology of endothelial cells in the lungs isolated from tamoxifen-injected TBRIca- Cdh5-Cre transgenic mice.

Expression of genes encoding extracellular matrix components in the skin and lungs of control and tamoxifen-injected TBRIca-Cdh5-Cre transgenic mice.

Expression of genes associated with myofibroblast differentiation in the skin and lungs of control and tamoxifen-injected TBRIca- Cdh5-Cre transgenic mice.

Expression of genes encoding profibrotic TGF-β-regulated transcription factors in the skin and lungs of control and tamoxifen-injected TBRIca-Cdh5-Cre transgenic mice.