Microangiopathy is a major cause of morbidity in type 2 diabetes mellitus (T2DM). While characteristic changes in blood capillary walls and endothelial dysfunction of blood vessels are well studied in type 2 diabetes, examination of lymphatic endothelial cells (LECs) and lymphatic vessels (LVs) is scarcely done. However, complications seen in type 2 diabetes, e.g. increased risk for infections, wound healing defects and obesity, may be related to lymphatic dysfunction. Therefore, we aimed at comprehensively analyzing potential morphological and structural differences of lymphatic endothelial cells and lymphatic vessels in the skin of type 2 diabetes mellitus patients. Further, we wanted to identify gene expression signatures that are deregulated in human dermal lymphatic vessels to define mechanisms that are linked with microvascular complications observed in type 2 diabetes.
By immunohistochemistry, basement membranes of lymph vessels were analyzed and blood and lymph vessel densities of diabetic versus normoglycemic skin was evaluated. Further, we identified signs of inflammation, e.g. macrophage infiltration and TNF alpha expression. We compared the gene expression profiles of ex vivo isolated dermal LECs retrieved from normoglycemic versus type 2 diabetic patients using microarrays and subsequent intensive bioinformatical analysis. The up- or downregulated expression of selected candidate genes was confirmed by quantitative real-time PCR and immunofluorescence stainings. Further, we focused on two differentially regulated genes and performed macrophage adhesion, transmigration and chemotatic assays as well as siRNA-mediated knockdown experiments to identify their specific function in lymphatic endothelial cells. Neither prominent alterations in extracellular matrix (ECM) protein deposition, nor morphological BM changes of lymphatic capillaries and collecting LVs were found in the skin of T2DM patients. This excluded the occurrence of diabetic lymphangiopathy comparable to that of blood vessels. However, the evaluation of lymph vessel counts revealed a prominent enhanced lymph vessel density in type 2 diabetic patient's skin. Further, we traced a strong macrophage infiltration in the dermis of type 2 diabetic patients. These macrophages produced vascular endothelial growth factors VEGF-A and VEGF-C, as well as the pro-inflammatory cytokine TNF alpha. Transcriptomal analysis of ex vivo isolated diabetic versus non-diabetic LECs resulted in a list of 180 differently expressed genes. Consistent with earlier studies, we identified several genes that have already been linked to genetic susceptibility for type 2 diabetes, including HP, APOD, HHEX, CD55, ANXA1, LMNA and FABP4. Essentially, we observed multiple changes related to altered LEC proliferation, adhesion and migration. Further, in line with increased TNF alpha abundance, we observed expression changes of CXCL10, VCAM1, CYR61, CXADR, SDC1 and AQP3. TNF alpha treatment of cultured LECs led to deregulated expression of selected genes, recapitulating the array results, indicating that TNF alpha is one major contributor to diabetes-specific gene expression signatures in lymphatic endothelial cells. CXCL10 was confirmed as one important candidate gene only expressed in chronically inflamed lymphatic vessels, contributing to adhesion and transmigration of macrophages and possibly intending to resolute the dermal inflammation. Further, the fatty acid transporter FABP4 was specifically upregulated in LECs and lymphatic vessels in type 2 diabetes in comparison to blood endothelial cells (BECs) and blood vessels. FABP4 was shown to regulate LEC proliferation and permeability in vitro, and pointed out the crucial role of lymphatic vessels in fatty acid transport and metabolism.
These data reveal gene sets highlighting the dramatically altered milieu skin lymphatic vessels have to cope with during type 2 diabetes mellitus. Further, we discovered that skin lymphatics show a chronic subacute inflammatory phenotype characterized by macrophage recruitment and de novo lymphangiogenesis. We provide evidence for a paracrine crosstalk, mainly via TNF alpha and CXCL10, fostering macrophage recruitment to LECs as one pathophysiological process that might contribute to persistent inflammation and consecutively, aberrant lymphangiogenesis in the skin.