Autophagy Inhibits Inflammation via Down-Regulation of p38 MAPK/mTOR Signaling Cascade in Endothelial Cells.

Objective: Autophagy, an intracellular process of self-digestion, has been shown to modulate inflammatory responses. In the present study, we determined the effects of autophagy on inflammatory response induced by M5 cytokines. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with M5 cytokines to induce inflammation. Expression levels of mRNA for inflammatory cytokines and BIRC2 were compared in HUVECs with vs without induction of autophagy with rapamycin (RAPA) by PCR, while cell apoptosis was assessed by flow cytometry and caspase-3 activity assay kit. Expression levels of LC3, p62, p-p38 MAPK (Thr180/Tyr182), p-mTOR (Ser2445) and p-ULK1 (Ser555) proteins were measured by Western blotting. The nitric oxide (NO) content, NO synthase (NOS) activity and cell angiogenesis were also evaluated. Results: Induction of autophagy with RAPA decreased expression levels of IL6, IL8 and CCL20, in addition to reduction in inflammation-induced apoptosis in HUVECs. Moreover, RAPA increased LC3II, while decreasing p62 expression. Likewise, expression levels of p-p38 MAPK and p-mTOR proteins were markedly decreased by the treatment with RAPA. Finally, RAPA treatment increased the NO content and the NOS activity, and inhibited angiogenesis. Conclusion: Induced autophagy can improve the function of endothelial cells in psoriasis, suggesting approaches to induce autophagy can be used to ameliorate psoriasis.

ITGA9 Inhibits Proliferation and Migration of Dermal Microvascular Endothelial Cells in Psoriasis.

Background: Cell proliferation, migration, and angiogenesis are aberrant in psoriatic human dermal microvascular endothelial cells (HDMECs), resulting in abnormal endothelial function and microvascular dilation in psoriasis. Objective: To explore the role of Integrin subunit alpha 9 (ITGA9) in proliferation and migration of dermal microvascular endothelial cells. Methods: HDMECs were isolated from the skin of 6 psoriatic patients and 6 healthy controls. Expression levels of ITGA9 mRNA and protein were assessed with qRT-PCR and Western blot, respectively, while miqRT-PCR was used to determine expression levels of miR-146a-3p. Cell proliferation and migration were assessed in human microvascular endothelial cell line (HMEC-1), following overexpression of either ITGA9 or miR-146a-3p, or co-transfection with miR-146a-3p-mimic and pLVX - ITGA9. Cell viability was detected by Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay. Cell apoptosis was assessed, using annexin V-FITC/PI apoptosis detection kit, while cell migration was detected by wound healing and transwell assay. Results: Expression levels of ITGA9 were significantly decreased in psoriatic HDMECs compared to normal controls. Moreover, expression levels of miR-146a-3p were higher in psoriatic HDMECs than in normal controls. Overexpression of miR-146a-3p lowered expression levels of ITGA9, accompanied by increased proliferation and migration of HMEC-1 in vitro. In contrast, overexpression of ITGA9 inhibited proliferation and migration of HMEC-1, while increasing expression levels of cdc42, ki67, focal adhesion kinase (FAK), c-Src tyrosine kinase (Src), RAC1 and RhoA. Conclusion: ITGA9 can repress the proliferation and migration of HMEC-1, suggesting utility of ITGA9 as a potential therapeutic intervention for psoriasis.

Immunomodulatory effect of psoriasis-derived dermal mesenchymal stem cells on TH1/TH17 cells.

T cell-mediated inflammation plays an important role in the development of psoriasis. Mesenchymal stem cells (MSCs) are a population of multipotent cells that regulate the T cell-mediated immune response. To investigate the effects of psoriatic dermal mesenchymal stem cells (p-DMSCs) on proliferation, apoptosis and differentiation of T cells. p-DMSCs and normal DMSCs (n-DMSCs) were isolated from psoriatic skin and normal healthy controls, respectively, and co-cultured with activated T cells isolated from healthy volunteers using a Transwell system. Proliferation and apoptosis of T cells were assessed by cell count and flow cytometry, respectively. Expression levels of transcription factors associated with subtypes of T cells and cytokines were measured by qRT-PCR and western blot. Both p-DMSCs and n-DMSCs inhibited T cell proliferation and cytokine production. Similarly, the presence of p-DMSCs and n-DMSCs decreased the expression levels of both T-bet and ROR-γt in T cells. However, n-DMSCs exhibited a stronger inhibitory effect than p-DMSCs on T cell proliferation, cytokine production, and T-bet and ROR-γt expression. These results suggest that the effect of p-DMSCs on T cell function could contribute, at least in part, to the pathogenesis of psoriasis.

Increased angiogenesis and migration of dermal microvascular endothelial cells from patients with psoriasis.

Psoriasis displays both increased angiogenesis and microvascular dilation in the skin, while human dermal microvascular endothelial cells (HDMECs) are involved in angiogenesis and microvascular dilation. Whether the functions of HDMECs are altered in psoriatic skin versus healthy skin remain unknown. Here, we isolated HDMECs from the skin of 10 patients with psoriasis and 10 healthy subjects and compared angiogenesis, proliferation, migration and cell metabolism between psoriatic HDMECs and normal HDMECs. We found that the morphology of primary HDMECs was comparable between psoriatic HDMECs and normal HDMECs. After passage, psoriatic HDMECs displayed larger cell size and wider intercellular space. In addition to DiI-Ac-LDL (DiI-labelled acetylated low-density lipoprotein) uptake, expression levels of CD31, vWF (von Willebrand factor) and LYVE-1 were comparable in psoriatic HDMECs versus normal HDMECs. However, psoriatic HDMECs exhibited increased tube formation (numbers of nodes and meshes, p < 0.05) and migration (numbers of migrated cells, p < 0.001) and reductions in proliferation (growth rates, p < 0.05) and energy metabolism (oxygen consumption rate and extracellular acidification rate, p < 0.05) compared with normal HDMECs. Therefore, psoriatic HDMECs display an increased angiogenesis and migration and decreased proliferation and metabolic activity, suggesting a pathogenic role of HDMECs in psoriasis.

Psoriatic mesenchymal stem cells stimulate the angiogenesis of human umbilical vein endothelial cells in vitro.

OBJECTIVE: To investigate the regulation of psoriatic dermal mesenchymal stem cells (p-DMSCs) in the expression of vascular growth factor (VEGF), and migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: A co-culture model of HUVECs and dermal mesenchymal stem cells (DMSCs)was used in this study. After 7-day co-culture, changes in expression levels of VEGF mRNA and protein in HUVECs were assessed using RT-PCR and Western Blotting, respectively. Migration and tubular formation of HUVECs were also assessed following co-culture of DMSCs and HUVECs. RESULTS: In comparison to either HUVECs alone or co-culture of n-DMSCs and HUVECs, co-culture of HUVECs and p-DMSCs significantly increased expression levels of both VEGF mRNA (p < 0.01 vs. HUVECs alone) and protein in HUVECs (p < 0.001 vs. both HUVECs alone and HUVECs co-cultured with n-DMSCs). Moreover, p-DMSCs stimulated HUVEC migration and vascular formation (p < 0.05 vs. both HUVECs alone and co-culture of n-DMSCs and HUVECs). CONCLUSION: Psoriatic DMSCs can upregulate VEGF expression, and stimulate migration and angiogenesis of HUVECs, suggesting a pathogenic role of p-DMSCs in psoriasis.

Dysregulated Dermal Mesenchymal Stem Cell Proliferation and Differentiation Interfered by Glucose Metabolism in Psoriasis.

BACKGROUND AND OBJECTIVES: Psoriasis is a chronic inflammatory skin disease, which the mechanisms behind its initiation and development are related to many factors. DMSCs (dermal mesenchymal stem cells) represent an important member of the skin microenvironment and play an important role in the surrounding environment and in neighbouring cells, but they are also affected by the microenvironment. We studied the glucose metabolism of DMSCs in psoriasis patients and a control group to reveal the relationship among glucose metabolism, cell proliferation activity，and VEC (vascular endothelial cell) differentiation in vitro, we demonstrated the biological activity and molecular mechanisms of DMSCs in psoriasis. METHODS AND RESULTS: We found that the OCR of DMSCs in psoriatic lesions was higher than that in the control group, and mRNA of GLUT1 and HK2 were up-regulated compared with the control group. The proliferative activity of DMSCs in psoriasis was reduced at an early stage, and mRNA involved in proliferation, JUNB and FOS were expressed at lower levels than those in the control group. The number of blood vessels in psoriatic lesions was significantly higher than that in the control group (p<0.05), which the mRNA of VEC differentiation, CXCL12, CXCR7, HEYL and RGS5 tended to be increased in psoriatic lesions compared to the control group, in addition to Notch3. CONCLUSIONS: We speculated that DMSCs affected local psoriatic blood vessels through glucose metabolism, and the differentiation of VECs, which resulted in the pathophysiological process of psoriasis.

An effective method of isolating microvascular endothelial cells from the human dermis.

Dermal microvascular endothelial cells (DMECs) play central roles in inflammation and angiogenesis and have become important cell models for studying various skin diseases. However, primary DMECs are difficult to culture and often contaminated by mesenchymal stem cells, fibroblasts, and other stromal cells. Surgically removed superfluous foreskin was first cut into pieces, digested with two types of enzymes, and dispersed into single cells. Cells obtained from the dermis were then subjected to Percoll density gradient centrifugation and cells located between densities 1.033 and 1.047 g/ml were further purified with endothelial growth medium containing decreasing concentrations of puromycin. Obtained HDMECs were identified by microscopy, flow cytometry, quantitative reverse-transcription polymerase chain reaction, western blot analysis, and immunofluorescent staining. The expression of CD31 (PECAM-1), CD34, VEGFR2, VWF (Von Willebrand Factor), VE-Cadherin (CD144), and NOS was positive. HDMECs were found to have abilities of angiogenesis and uptake of acetylated low-density lipoprotein. Growth curves and cell viability were analyzed, and a growth pattern consisting of the "latency phase-logarithmic growth phase-stagnation phase" was determined. In this study, a simple, rapid, effective, and low-cost method is established to isolate HDMECs from the foreskin with a purity of over 91% and high viability. The method showed good repeatability and allowed a stable passage. This study provides technical support and theoretical guidance for studying the physiological characteristics of HDMECs, the pathogenesis of the skin associated, and other microvascular diseases.

Multi-omics study in monozygotic twins confirm the contribution of de novo mutation to psoriasis.

BACKGROUND: Genome-wide association studies have identified over 120 risk loci for psoriasis. However, most of the variations are located in non-coding region with high frequency and small effect size. Pathogenetic variants are rarely reported except HLA-C*0602 with the odds ratio being approximately 4.0 in Chinese population. Although rare variations still account for a small proportion of phenotypic variances in complex diseases, their effect on phenotypes is large. Recently, more and more studies focus on the low-frequency functional variants and have achieved a certain amount of success. METHOD: Whole genome sequencing and sanger sequencing was performed on 8 MZ twin pairs discordant for psoriasis to scan and verified the de novo mutations (DNMs). Additionally, 665 individuals with about 20 years' medical history versus 2054 healthy controls and two published large population studies which had about 8 years' medical history (including 10,727 cases versus 10,582 controls) were applied to validate the enrichment of rare damaging mutations in two DNMs genes. Besides, to verify the pathogenicity of candidate DNM in C3, RNA-sequencing for CD4+, CD8+ T cells of twins and lesion, non-lesion skin of psoriasis patients were carried out. Meanwhile, the enzyme-linked immunosorbent assay kit was used to detect the level of C3, C3b in the supernatant of peripheral blood. RESULT: A total of 27 DNMs between co-twins were identified. We found six of eight twins carry HLA-C∗0602 allele which have large effects on psoriasis. And it is interesting that a missense mutation in SPRED1 and a splice region mutation in C3 are found in the psoriasis individuals in the other two MZ twin pairs without carrying HLA-C*0602 allele. In the replication stage, we found 2 loss-of-function (LOF) variants of C3 only in 665 cases with about 20 years' medical history and gene-wise analysis in 665 cases and 2054 controls showed that the rare missense mutations in C3 were enriched in cases (OR = 1.91, P = 0.0028). We further scanned the LOF mutations of C3 in two published studies (about 8 years' medical history), and found one LOF mutation in the case without carrying HLA-C*0602. In the individual with DNM in C3, RNA sequencing showed the expression level of C3 in skin was significant higher than healthy samples in public database (TPM fold change = 1.40, P = 0.000181) and ELISA showed protein C3 in peripheral blood was higher (~2.2-fold difference) than the other samples of twins without DNM in C3. CONCLUSION: To the best of our knowledge, this is the first report that DNM in C3 is the likely pathological mutations, and it provided a better understanding of the genetic etiology of psoriasis and additional treatments for this disease.

Efficient differentiation of vascular endothelial cells from dermal-derived mesenchymal stem cells induced by endothelial cell lines conditioned medium.

OBJECTIVE: To directionally-differentiate dermis-derived mesenchymal stem cells (DMSCs) into vascular endothelial cells (VECs) in vitro, providing an experimental basis for studies on the pathogenesis and treatment of vascular diseases. METHODS: After separation by adherent culture, VEC line supernatant, vascular endothelial growth factor (VEGF), bone morphogenetic protein-4 and hypoxia were used for the differentiation of VECs from DMSCs. The cell type was authenticated by flow cytometry, matrigel angiogenesis assay in vitro, and immunofluorescent staining during differentiation. The VEGF concentration was investigated by enzyme-linked immunosorbent assay. RESULTS: After 28 days of differentiation, the cell surface marker CD31 was significantly positive (80%-90%) by flow cytometry in the VEC line-conditioned culture, which was significantly higher than in the other groups. Differentiated DMSCs had the ability to ingest Dil-ac-LDL and vascularize in the conditioned culture, but not in the other groups. In the VEC line supernatant, the concentration of VEGF was very low. The VEGF concentration changed along with the differentiation into VECs in the medium of the conditioned culture group. CONCLUSION: VEC line supernatant can induce the differentiation of DMSCs into VECs, possibly through the pathway except VEGF.