Exploring Cardiac Microvasculature Injury Markers CFH And Target Intervention Point Ets1 In Diabetes Based On Single Cell RNA Sequencing

Background:Diabetes is a pressing public health issue of contemporary society,posing significant threats to human health.Cardiac microvascular damage is a prevalent complication amongst diabetic patients and a significant contributor to mortality in this patient demographic.Endothelial cells are the fundamental constituents of blood vessels,not only regulating material exchange between blood and tissue fluid but also secreting diverse cytokines,thereby possessing crucial physiological functions.Endothelial dysfunction is a salient feature of diabetic cardiac microvascular injury,with extensive reports on its pathological mechanisms available from prior studies;however,the heterogeneity of endothelial cells has remained unresolved.Traditional RNA sequencing techniques often confine themselves to average gene expression levels,obscuring cellular heterogeneity and potential alterations.Single cell RNA sequencing technology can identify the identity,function,and state of individual cells,thus providing precise information about intricate cellular activities within organisms.Moreover,current methods for evaluating cardiac microvascular function encompass noninvasive examinations and interventional procedures,yet these diagnostic modalities bear high medical costs,and there remains a lack of convenient and effective biomarkers for diabetic cardiac microvascular injury.It is particularly imperative to explore the critical mechanisms underlying diabetic microvascular endothelial injury using single cell sequencing technology and accurately predict clinical risks associated with diabetes.Aims:1.To identify biological markers and therapeutic targets for diabetic cardiac microvascular injury through single cell RNA sequencing;2.To evaluate the predictive value of CFH,an identified potential marker,for diabetic cardiac microvascular injury via clinical trials;3.To investigate the role of Ets1,an identified potential target,in diabetic cardiac microvascular injury through basic experiments.Methods:1.The diabetic model was established using leptin receptor knockout mice(db/db),subsequently isolating single cells from the hearts of db/db mice at various pathological stages and heterozygous control mice(db/m).Following rigorous data quality control,cell type annotation,and dynamic,fate,and functional analysis,key cellular subpopulations were identified.Transcription factor analysis was employed to elucidate the critical regulatory mechanisms underlying cardiac microvascular injury in diabetes.2.The alterations in potential markers and intervention targets within cardiac microvessels of db/db mice across different pathological stages were examined through immunofluorescence.Additionally,the fluctuations of potential markers in plasma among db/db mice at various disease states were validated via immunohistochemistry.3.Employing the two independent sample t test or Mann-Whitney U test to assess potential disparities in coronary flow reserve(CFR),microcirculation resistance index(IMR),potential biomarkers,and control markers(s VE-cadherin and Endocan)between patients with diabetes and non-diabetic individuals within a clinical sample.Utilizing Pearson correlation analysis to evaluate the associations of these plasma markers with indices of microvascular function(CFR and IMR).Lastly,utilizing receiver operating characteristic(ROC)curves to gauge the diagnostic efficacy of these plasma markers for cardiac microvascular injury in diabetic patients.4.The endothelium-specific Adeno Associated Virus(AAV)was employed to interfere the expression level of potential targets in db/db mice and db/m mice.Following successful overexpression of cardiac vascular potential target,vessel density was assessed through CD31 staining;barrier function of blood vessels was evaluated by transmission electron microscopy for basal membrane thickness and VE-cadherin staining;perivascular fibrosis was quantified using Masson’s trichrome stain;and cardiac function was evaluated via echocardiography.Results:1.Following quality assurance and data refinement,a total of 63,825 cells were procured from the hearts of eight cohorts of mice with varying stages of diabetes,which were compartmentalized into 37 clusters,encompassing fibroblasts,endothelial cells,and an insignificant proportion of cardiomyocytes and immune cells.There existed eleven clusters of endothelial cells,specifically 3,5,10,12,15,20,21,22,26,31,34 clusters.2.Pseudo-time series analysis concluded that cluster 12 may represent the terminal state of endothelial cell differentiation in diabetic cardiac microvascular injury,primarily functioning as a mediator for inflammatory cell penetration into the vascular wall.Variation in inter-group subpopulation ratios suggests a progressive increase in the proportion of cluster 12 with advancing diabetes mellitus,representing the highest proportion of endothelial cell subsets at late stages of diabetes,while CFH emerges as the most prominent characteristic gene for cluster 12.3.SCENIC transcription factor examination identified Ets1 as the primary regulatory factor in controlling endothelial cells during diabetic injury.The Ets1 regulatory network displays substantial relation to angiogenesis but demonstrated reduced regulatory engagement during diabetic heart microvascular damage.4.The results from the clinical investigation demonstrate a statistically significant augmentation in plasma CFH concentrations in patients afflicted with diabetes when contrasted with non-diabetic individuals.Simultaneously,within the diabetic cohort,there exists a robust correlation between plasma CFH levels and microvascular function parameters(CFR and IMR).Employing ROC curve to evaluate the diagnostic capability of plasma CFH levels for cardiac microvascular injury in diabetics,the findings suggest an area under the curve(AUC)of 0.893(IMR≥25)and 0.851(CFR<2),indicating that plasma CFH levels exhibit superior diagnostic capability for cardiac microvascular injury in diabetics.5.Basic experimental results suggested that AAV9-Ets1 notably elevated cardiac vascular density,improved cardiac performance and vascular barrier function in db/db mice,declining plasma marker CFH levels and enhancing survival.Conclusions:Combined with single-cell RNA sequencing technology and related clinical trials and basic experiments,we found that plasma CFH is expected to become a specific marker of diabetic cardiac microvascular injury,and Ets1 overexpression is expected to become a new therapeutic strategy for diabetic cardiac microvascular injury.