Altered Properties of Endothelial Cells and Mesenchymal Stem Cells Underlying the Development of Scleroderma‐like Vasculopathy in KLF5^+/−;Fli‐1^+/− Mice

Abstract

Objective In prevous studies, we established a new animal model, KLF5+/−;Fli‐1+/− mice, in which fundamental pathologic features of systemic sclerosis (SSc) are broadly recapitulated. SSc vasculopathy is believed to occur as a result of impaired vascular remodeling, but its detailed mechanism of action remains unknown. To address this, the present study investigated the properties of dermal microvascular endothelial cells (DMECs), bone marrow–derived endothelial progenitor cells (BM‐EPCs), and bone marrow–derived mesenchymal stem cells (BM‐MSCs), a precursor of pericytes, in KLF5+/−;Fli‐1+/− mice. Methods Neovascularization and angiogenesis were assessed in KLF5+/−;Fli‐1+/− mice by in vivo Matrigel plug assay and in vitro tube formation assay, respectively. The properties of mouse BM‐EPCs and BM‐MSCs were assessed with in vitro studies. Dermal vasculature was visualized in vivo by injecting the mice with fluorescein isothiocyanate–conjugated dextran. Results Neovascularization was diminished in skin‐embedded Matrigel plugs from KLF5+/−;Fli‐1+/− mice. DMECs from KLF5+/−;Fli‐1+/− mice showed defective tubulogenic activity, decreased expression of VE‐cadherin and CD31, and an imbalance in the expression of Notch1/Dll4, suggesting that angiogenesis and anastomosis are disturbed. KLF5+/−;Fli‐1+/− mouse BM‐MSCs exhibited enhanced proliferation and migration and increased collagen production following stimulation with transforming growth factor β1, indicating that these cells differentiate preferentially into myofibroblasts rather than pericytes. KLF5+/−;Fli‐1+/− mouse BM‐EPCs displayed a transition toward mesenchymal cells, suggesting that vasculogenesis is impaired. Wound healing was delayed in KLF5+/−;Fli‐1+/− mice (mean ± SD healing time 15.67 ± 0.82 days versus 13.50 ± 0.84 days; P = 0.0017), and the vascular network was poorly developed in wound scar tissue. Conclusion The characteristics observed in the KLF5+/−;Fli‐1+/− mouse model — specifically, impaired neovascularization and vascular maturation — are similar to those observed in human SSc, and could be at least partially attributable to the induction of SSc‐like properties in DMECs, BM‐EPCs, and BM‐MSCs. These findings indicate the critical contribution of Klf5 and Fli1 deficiency in vascular cells and related cell precursors to the development of SSc vasculopathy.

New Insights into Profibrotic Myofibroblast Formation in Systemic Sclerosis: When the Vascular Wall Becomes the Enemy

“…Moreover, Fli1 haploinsufficiency has been found to induce a profibrotic phenotype in dermal ECs of bleomycin-treated mice, further strengthening the involvement of this transcription factor in EndoMT during the development of cutaneous fibrosis [ 57 , 58 ]. Recently, dermal ECs explanted from KLF5+/−; Fli1+/− mice, a new animal model resembling the fundamental pathologic features of SSc, were reported not only to be defective in performing in vitro angiogenesis, but also to have a reduced expression of VE-cadherin and CD31, suggesting the occurrence of EndoMT, as observed in SSc-affected ECs, and highlighting that a deficiency of KLF5 transcription factor may be another important trigger of this process [ 59 ]. SSc-related EndoMT has been recently found to be induced also by oncostatin M, a member of the IL-6 family, and the inflammatory lipid mediator leukotriene B4, whose expression levels are increased in SSc [ 60 , 61 ].…”

“…Furthermore, prolonged exposure of BM-MSCs to a profibrotic milieu mimicking the SSc microenvironment increased the tendency of these cells to differentiate into myofibroblasts [ 98 ]. Finally, similarly to human SSc BM-MSCs, it has been reported that also BM-MSCs from the KLF5+/−; Fli1+/− murine model of SSc display enhanced migration, proliferation and collagen production in response to TGF-β1, thus showing a preferential differentiation toward myofibroblasts instead of behaving as pericyte precursors [ 59 ].…”

Endothelial-to-mesenchymal transition in systemic sclerosis

“…Recently, in an experimental model of SSc, the double heterozygous mice for Klf5 and Fli1 (Klf5 +/− ; Fli1 +/− ), showing the three main pathological characteristics of SSc [159], the dermal microvascular ECs isolated from these mice, show defective angiogenesis and reduced expression of VE‐cadherin and CD31, confirming the evidence of EndMT in this SSc model [160].…”

Endothelial CCR6 expression due to FLI1 deficiency contributes to vasculopathy associated with systemic sclerosis

“…These vascular alterations are caused by dysregulated angiogenesis and defective vasculogenesis. FLI1 deficiency is a key disease factor regulating a broad spectrum of endothelial behaviors and vascular remodeling associated with SSc vasculopathy, including angiogenesis and vasculogenesis [ 45 ]. FLI1 deficiency suppresses the expression of CD31, VE-cadherin, S1P 1, and platelet-derived growth factor-B in endothelial cells, while upregulating matrix metalloproteinase-9, resulting in vascular destabilization and angiogenesis [ 29 ].…”