losartan-potassium and Vascular-Calcification

The current research aimed to verify the effects of erythropoietin (EPO) on vascular calcification under inflammatory conditions and the molecular regulator of vascular calcification induced by EPO. To induce vascular calcification and systemic chronic inflammation in SD rats, EPO was administered intraperitoneally, and 10% casein was injected subcutaneously. The administration period lasted for 20 consecutive weeks. Blood samples were subsequently collected to detect inflammatory factors and vascular calcification. Additionally, high-dose EPOs were applied to stimulate primary vascular smooth muscle cells (VSMCs), and vascular calcification was measured using alizarin red staining, alkaline phosphatase (ALP) activity, and calcium salt quantification. The probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) was employed to detect cellular reactive oxygen species (ROS) levels. The expressions of bone formation-related protein and anti-calcification protein matrix gla protein (MGP) were determined via Western blot. Compared with the control group, calcium deposits and vascular calcification were increased in the EPO group, tumor necrosis factor-alpha (TNF-α) group and TNF-α+ EPO group, whereas MGP was significantly reduced. Moreover, under the stimulation of TNF-α and EPO+TNF-α, pp38/p38 was increased substantially, the addition of p38 inhibitor SB203580 could significantly reduce calcium deposits and vascular calcification. In vivo experiment, compared with the EPO group, calcium salt deposition and vascular calcification were elevated in the EPO+casein group. The present results revealed that high-dose EPO could cause calcification of the abdominal aorta in rats. The inflammatory response aggravated the vascular calcification induced by EPO via activating p38 and ROS levels.

Topics: Animals; Calcium; Caseins; Cells, Cultured; Erythropoietin; Inflammation; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Tumor Necrosis Factor-alpha; Vascular Calcification

To investigate its effect and molecular regulatory mechanism on vascular calcification, EPO was added to vascular smooth muscle cells cultured in vitro and injected intraperitoneally into SD rats. The effect of EPO on VSMC calcification was determined by alizarin red staining and ALP activity. Differentially expressed genes were screened by transcriptome sequencing and the relationship and function were verified. We found EPO promotes VSMC calcification in vitro and blood calcification in vivo in a dose-dependent manner. A total of 88 upregulated genes and 59 downregulated genes were detected in transcriptome sequencing, among which the expression of genes associated with bone formation exhibited a marked increase, namely the GATA6 transcription factor, BMP2, RUNX2, OPN, and OCN. Dual luciferase assay has indicated that the binding of GATA6 to BMP2 promoter facilitates the transcription of BMP2. Taken together, findings indicate that EPO can enhance the calcification of VSMCs by activating the GATA6/BMP2 signal axis.

Topics: Animals; Bone Morphogenetic Protein 2; Cells, Cultured; Erythropoietin; GATA6 Transcription Factor; Gene Expression Regulation; Injections, Intraperitoneal; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteogenesis; Rats, Sprague-Dawley; Signal Transduction; Transcriptome; Vascular Calcification

Previous studies suggested that endoplasmic reticulum (ER) stress induced-apoptosis promoted vascular calcification (VC). Interestingly, erythropoietin (EPO), an endogenous glycoprotein, exerts multiple tissue protective effects by inhibiting ER stress and apoptosis. We investigated the role and potential mechanism of EPO on VC in chronic kidney disease (CKD) rats and cultured vascular smooth muscle cells (VSMCs). The calcification model was established by subtotal nephrectomy in vivo or phosphate overload in vitro. The protein level of EPO receptor (EPOR) was increased in the calcified aortas of CKD rats. EPO prevented the reduction of VSMC phenotypic markers, and reversed the increased calcium content and calcium salt deposition in the aortas of CKD rats and cultured calcified VSMCs. The protein levels of activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94) were upregulated in aortas and VSMCs under calcifying conditions, indicating ER stress activation. EPO treatment of CKD rats or calcified VSMCs downregulated the protein levels of ATF4 and GRP94. Furthermore, ER stress-mediated apoptosis, determined by the protein levels of CCAAT⁄enhancer-binding protein-homologous protein and cleaved caspase 12, was increased in tunicamycin or calcification media-treated VSMCs, but the increased effect was reversed in EPO-treated groups. The increased apoptotic cells in calcified VSMCs, as indicated by Hoechst staining and flow cytometry, were downregulated by the co-administration of EPO or 4-phenyl butyric acid. In conclusion, EPO might attenuate VC by inhibiting ER stress mediated apoptosis through EPOR signaling.

Topics: Activating Transcription Factor 4; Animals; Aorta; Cells, Cultured; Endoplasmic Reticulum Stress; Erythropoietin; Male; Membrane Glycoproteins; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Vascular Calcification

Vascular calcification is common in chronic kidney disease (CKD) patients, while erythropoietin (EPO) is widely used in the treatment of renal anemia in CKD patients, whether there is a link between the two is still not clear.. The primary rat vascular smooth muscle cells (VSMCs) and CKD rats were treated with EPO and the calcium deposition was observed by alizarin red staining, von Kossa staining and calcium quantification. Activation of JAK2/STAT3/BMP-2 axis and NF-κB signaling pathways was investigated by Western blotting.. EPO-induced calcium deposition in VSMCs and significantly potentiated calcification in CKD rats. Furthermore, EPO activated JAK2/STAT3/BMP-2 axis, NF-κB pathway and the pro-calcification effect of EPO was partially blocked by the STAT3 inhibitor (Cryptotanshinone) or NF-κB inhibitor (BAY 11-7082), respectively, in vitro.. EPO could promote VSMCs calcification in vitro and in vivo and this effect may be achieved through the JAK2/STAT3/BMP-2 axis and NF-κB pathway.

Topics: Alkaline Phosphatase; Anemia; Animals; Bone Morphogenetic Protein 2; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Erythropoietin; Gene Expression; Janus Kinase 2; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Nitriles; Phenanthrenes; Phosphorylation; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Signal Transduction; STAT3 Transcription Factor; Sulfones; Up-Regulation; Vascular Calcification