Evaluating differential expression of fibrosis-related genes and their correlation with blood vessel density in chronic cutaneous graft-versus-host disease.

BACKGROUND: Sclerodermoid graft-versus-host disease (GVHD) is the most severe form of chronic GVHD (cGVHD) and represents a considerable therapeutic challenge. Due to the scarcity of human studies on sclerodermoid cGVHD, the pathogenesis of this entity is not fully understood. OBJECTIVE: To identify the differential expression of fibrosis-related genes in skin lesions of human lichenoid and sclerodermoid cGVHD and to assess the expression of their corresponding proteins. METHODS: PCR array analysis was performed on RNA extracted from three skin biopsies of sclerodermoid cGVHD patients and three normal skin samples, for fibrosis-related gene expression profiles followed by evaluation of their corresponding protein expressions. The expressions of Tissue inhibitor of metalloproteinase 3 (TIMP3), matrix metalloproteinase 1 (MMP1), TIMP1, and TIMP2 were further studied by immunohistochemistry. Demographic, clinical and immunohistochemical parameters of the two cGVHD groups and the control group were compared. The Pearson correlation coefficient was used to assess the correlation between data among the study groups. RESULTS: We identified 44 upregulated and 14 downregulated genes in the skin samples of sclerodermoid cGVHD compared to the control group. TIMP3 was positive in 13/21 biopsies of cGVHD and in one biopsy of the control group. The average staining intensity was significantly higher in the cGVHD group compared to the control group. TIMP3 was expressed mainly in dermal blood vessels. cGVHD specimens with positive TIMP3 staining had a statistically significantly higher total microvascular area than the negative specimens. CONCLUSION: TIMP3 levels are increased in both subtypes of cGVHD and are associated with increased dermal vascularity.

Successful Introduction of Human Renovascular Units into the Mammalian Kidney.

BACKGROUND: Cell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in in vivo formation of vascular networks. METHODS: We administered renal tubule-forming cells derived from human adult and fetal kidneys (previously shown to exert a functional effect in CKD mice) into mice, alongside MSCs and ECFCs. We then assessed whether this would result in generation of "renovascular units" comprising both vessels and tubules with potential interaction. RESULTS: Directly injecting vessel-forming cells and renal tubule-forming cells into the subcutaneous and subrenal capsular space resulted in self-organization of donor-derived vascular networks that connected to host vasculature, alongside renal tubules comprising tubular epithelia of different nephron segments. Vessels derived from MSCs and ECFCs augmented in vivo tubulogenesis by the renal tubule-forming cells. In vitro coculture experiments showed that MSCs and ECFCs induced self-renewal and genes associated with mesenchymal-epithelial transition in renal tubule-forming cells, indicating paracrine effects. Notably, after renal injury, renal tubule-forming cells and vessel-forming cells infused into the renal artery did not penetrate the renal vascular network to generate vessels; only administering them into the kidney parenchyma resulted in similar generation of human renovascular units in vivo. CONCLUSIONS: Combined cell therapy of vessel-forming cells and renal tubule-forming cells aimed at alleviating renal hypoxia and enhancing tubulogenesis holds promise as the basis for new renal regenerative therapies.

The role of the vasculature niche on insulin-producing cells generated by transdifferentiation of adult human liver cells.

BACKGROUND: Insulin-dependent diabetes is a multifactorial disorder that could be theoretically cured by functional pancreatic islets and insulin-producing cell (IPC) implantation. Regenerative medicine approaches include the potential for growing tissues and organs in the laboratory and transplanting them when the body cannot heal itself. However, several obstacles remain to be overcome in order to bring regenerative medicine approach for diabetes closer to its clinical implementation; the cells generated in vitro are typically of heterogenic and immature nature and the site of implantation should be readily vascularized for the implanted cells to survive in vivo. The present study addresses these two limitations by analyzing the effect of co-implanting IPCs with vasculature promoting cells in an accessible site such as subcutaneous. Secondly, it analyzes the effects of reconstituting the in vivo environment in vitro on the maturation and function of insulin-producing cells. METHODS: IPCs that are generated by the transdifferentiation of human liver cells are exposed to the paracrine effects of endothelial colony-forming cells (ECFCs) and human bone marrow mesenchymal stem cells (MSCs), which are the "building blocks" of the blood vessels. The role of the vasculature on IPC function is analyzed upon subcutaneous implantation in vivo in immune-deficient rodents. The paracrine effects of vasculature on IPC maturation are analyzed in culture. RESULTS: Co-implantation of MSCs and ECFCs with IPCs led to doubling the survival rates and a threefold increase in insulin production, in vivo. ECFC and MSC co-culture as well as conditioned media of co-cultures resulted in a significant increased expression of pancreatic-specific genes and an increase in glucose-regulated insulin secretion, compared with IPCs alone. Mechanistically, we demonstrate that ECFC and MSC co-culture increases the expression of CTGF and ACTIVINßα, which play a key role in pancreatic differentiation. CONCLUSIONS: Vasculature is an important player in generating regenerative medicine approaches for diabetes. Vasculature displays a paracrine effect on the maturation of insulin-producing cells and their survival upon implantation. The reconstitution of the in vivo niche is expected to promote the liver-to-pancreas transdifferentiation and bringing this cell therapy approach closer to its clinical implementation.

Somatic NRAS mutation in patient with generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA or lymphangiomatosis) is a rare disease characterized by a diffuse proliferation of lymphatic vessels in skin and internal organs. It often leads to progressive respiratory failure and death, but its etiology is unknown. Here, we isolated lymphangiomatosis endothelial cells from GLA tissue. These cells were characterized by high proliferation and survival rates, but displayed impaired capacities for migration and tube formation. We employed whole exome sequencing to search for disease-causing genes and identified a somatic mutation in NRAS. We used mouse and zebrafish model systems to initially evaluate the role of this mutation in the development of the lymphatic system, and we studied the effect of drugs blocking the downstream effectors, mTOR and ERK, on this disease.