Biology subjects

Rafikov, R.

Publications and source records attributed to Rafikov, R..

3 recordsLinked to original sources

bioRxiv2026

Efficient Isolation and Phenotypic Characterization of Primary Rat Pulmonary Pericytes

Pericytes are essential regulators of pulmonary vascular homeostasis, but their isolation from rat lung tissue is challenging because no single marker uniquely identifies them and contaminating fibroblasts, endothelial cells, hematopoietic cells, and vascular smooth muscle cells can persist during isolation and culture. Here, we describe a rapid and efficient protocol for the isolation of primary rat pulmonary pericytes using sequential magnetic depletion of CD45-positive hematopoietic cells and CD31-positive endothelial cells, followed by positive selection for NG2-positive cells. The isolated cells were expanded in culture and characterized by immunofluorescence and functional co-culture assays. Cultured cells displayed typical pericyte morphology and expressed the pericyte-associated markers NG2, PDGFR{beta}, and 3G5, with minimal expression of CD31, CD45, PDGFR and MYH11. In endothelial cell-pericyte ECM gel co-culture assays, isolated pericytes associated with endothelial cords and were frequently observed near network branch points and junctions, further supporting their pericyte identity. This method yields an enriched population of primary pulmonary pericytes suitable for downstream applications, including cell culture and functional studies. Overall, this streamlined protocol provides a practical platform for studying pulmonary pericyte biology in rat models of health and vascular disease.

cell biology↗

bioRxiv2025

Akt1 Nitration Promotes Proliferation and Mesenchymal Transition Exacerbating Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vascular remodeling and increased pulmonary arterial resistance. This study investigates the role of Akt1 nitration in PAH development, focusing on its effect on endothelial-to-mesenchymal transition (EndMT) and vascular cell proliferation. Using the novel Akt1Y350F mutant mouse model, which resists nitration due to a tyrosine-to-phenylalanine substitution, we demonstrated that Akt1 nitration is a key pathogenic factor in PAH progression. Our results show that Akt1Y350F mice, resistant to Akt1 nitration, are protected against PAH in a SU5416/hypoxia (SU5416/Hx) model, exhibiting lower right ventricular systolic pressure (RVSP), reduced right ventricular hypertrophy, and decreased vascular occlusion. Additionally, we identified important molecular mechanisms involving TWIST1, SMA, HIF1, and STAT3 signaling pathways that influence EndMT and vascular remodeling. The proteomic analysis revealed other affected pathways, including angiogenesis, lipid metabolism, and mitochondrial function. We demonstrate that oxidative and nitrative stress-induced post-translational modifications contribute to the pathological processes leading to pulmonary hypertension, using a unique Akt nitration-resistant mouse model. These findings provide new insights into the molecular mechanisms underlying PAH and suggest that targeting Akt1 nitration could be a promising therapeutic approach for this devastating disease.

physiology↗

bioRxiv2024

Endothelial fatty acid-binding proteins contribute to the pathogenesis of pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Single-cell RNA sequencing (scRNAseq) analysis found that both FABP4 and FABP5 were highly induced in endothelial cells (ECs) of Egln1Tie2Cre (CKO) mice, which was also observed in pulmonary arterial ECs (PAECs) from idiopathic PAH (IPAH) patients, and in whole lungs of pulmonary hypertension (PH) rats. Plasma levels of FABP4/5 were upregulated in IPAH patients and directly correlated with severity of hemodynamics and biochemical parameters using plasma proteome analysis. Genetic deletion of both Fabp4 and 5 in CKO mice (Egln1Tie2Cre/Fabp4-5-/-, TKO) caused a reduction of right ventricular systolic pressure (RVSP) and RV hypertrophy, attenuated pulmonary vascular remodeling and prevented the right heart failure assessed by echocardiography, hemodynamic and histological analysis. Employing bulk RNA-seq and scRNA-seq, and spatial transcriptomic analysis, we showed that Fabp4/5 deletion also inhibited EC glycolysis and distal arterial programming, reduced ROS and HIF-2 expression in PH lungs. Thus, PH causes aberrant expression of FABP4/5 in pulmonary ECs which leads to enhanced ECs glycolysis and distal arterial programming, contributing to the accumulation of arterial ECs and vascular remodeling and exacerbating the disease.

molecular biology↗