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Impedimetric Detection and Electromediated Apoptosis of Vascular Smooth Muscle Using Microfabricated Biosensors for Diagnosis and Therapeutic Intervention in Cardiovascular Diseases

ABSTRACT: Abstract Cardiovascular diseases remain a significant global burden with 1?in?3 of all deaths attributable to the consequences of the disease. The main cause is blocked arteries which often remain undetected. Implantable medical devices (IMDs) such as stents and grafts are often used to reopen vessels but over time these too will re?block. A vascular biosensor is developed that can report on cellularity and is amenable to being mounted on a stent or graft for remote reporting. Moreover, the device is designed to also receive currents that can induce a controlled form of cell death, apoptosis. A combined diagnostic and therapeutic biosensor would be transformational for the treatment of vascular diseases such as atherosclerosis and central line access. In this work, a cell sensing and cell apoptosing system based on the same interdigitated electrodes (IDEs) is developed. It is shown that the device is scalable and that by miniaturizing the IDEs, the detection sensitivity is increased. Apoptosis of vascular smooth muscle cells is monitored using continuous impedance measurements at a frequency of 10 kHz and rates of cell death are tracked using fluorescent dyes and live cell imaging. Vascular stenosis is a major complication of cardiovascular pathologies such as heart attack, stroke, and failed vascular access. An implantable self?reporting biosensor that detects the pathological changes offers a diagnostic early warning system. Importantly the sensor has dual function and is able to induce a therapeutic and controlled form of cell death. The design is scalable, with detection sensitivity increasing with miniaturization.

SUBMITTER: Bussooa A

PROVIDER: S-EPMC7509665 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Project description:Background and Aims: Atherosclerosis, a major contributor to cardiovascular and cerebrovascular diseases, remains a leading cause of global mortality and morbidity. This study focuses on the role of chemokine-like receptor 1 (CMKLR1) in VSMCs and its potential impact on atherosclerotic plaque formation and remodelling. Methods: Cmklr1-/-Apoe-/- mice, generated using CRISPR/Cas9 technology, were fed a high-fat western diet for 16 weeks to model atherosclerosis. Primary VSMCs were isolated from Cmklr1-/- and Cmklr1+/+ mice for in vitro experiments. Cell viability, senescence-associated β-galactosidase staining, and RNA interference were performed to assess the effect of CMKLR1 deficiency on VSMCs. Various assays, including flow cytometry, immunocytochemistry, and RNA-seq analysis, were employed to investigate the molecular mechanisms and downstream signals associated with CMKLR1 deficiency. Results: CMKLR1 deficiency was found to disrupt the cell cycle, leading to VSMC senescence both in vitro and in vivo. Cmklr1-/-Apoe-/- mice exhibited increased atherosclerotic plaque burden, emphasizing the role of CMKLR1 in plaque stability. Transcriptome analysis revealed significant changes in cellular components and biological processes, with a particular impact on the extracellular matrix and cellular response to stimulus. The TGFβ signalling pathway emerged as a potential downstream target of CMKLR1, affecting VSMC differentiation, vessel development, and extracellular matrix synthesis. The deficiency of CMKLR1 led to a reduction in TGFβ1 expression and downstream molecule SM22α, influencing plaque composition and stability. Conclusion: This study demonstrates that CMKLR1 deficiency promotes VSMC senescence and exacerbates atherosclerosis. The disrupted TGFβ signalling pathway, identified as a potential molecular mechanism, provides insights into the complex regulatory network associated with CMKLR1 in atherosclerotic plaque formation and remodelling. These findings highlight the importance of CMKLR1 in atherosclerosis and its potential as a therapeutic target for plaque stability.

Project description:BackgroundAbnormal proliferation of vascular smooth muscle cells (VSMC) is a major cause of cardiovascular diseases (CVDs). Many studies suggest that vascular injury triggers VSMC dedifferentiation, which results in VSMC changes from a contractile to a synthetic phenotype; however, the underlying molecular mechanisms are still unclear.MethodsIn this study, we examined how VSMC responds under mechanical stress by using time-course microarray data. A three-phase study was proposed to investigate the stress-induced differentially expressed genes (DEGs) in VSMC. First, DEGs were identified by using the moderated t-statistics test. Second, more DEGs were inferred by using the Gaussian Graphical Model (GGM). Finally, the topological parameters-based method and cluster analysis approach were employed to predict the last batch of DEGs. To identify the potential drugs for vascular diseases involve VSMC proliferation, the drug-gene interaction database, Connectivity Map (cMap) was employed. Success of the predictions were determined using in-vitro data, i.e. MTT and clonogenic assay.ResultsBased on the differential expression calculation, at least 23 DEGs were found, and the findings were qualified by previous studies on VSMC. The results of gene set enrichment analysis indicated that the most often found enriched biological processes are cell-cycle-related processes. Furthermore, more stress-induced genes, well supported by literature, were found by applying graph theory to the gene association network (GAN). Finally, we showed that by processing the cMap input queries with a cluster algorithm, we achieved a substantial increase in the number of potential drugs with experimental IC50 measurements. With this novel approach, we have not only successfully identified the DEGs, but also improved the DEGs prediction by performing the topological and cluster analysis. Moreover, the findings are remarkably validated and in line with the literature. Furthermore, the cMap and DrugBank resources were used to identify potential drugs and targeted genes for vascular diseases involve VSMC proliferation. Our findings are supported by in-vitro experimental IC50, binding activity data and clinical trials.ConclusionThis study provides a systematic strategy to discover potential drugs and target genes, by which we hope to shed light on the treatments of VSMC proliferation associated diseases.

Project description:AimsQuiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here, we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality.Methods and resultsWe investigate the dynamics of VSMC clone formation using confocal microscopy and single-cell transcriptomics in VSMC-lineage-traced animal models. We find that activation of medial VSMC proliferation occurs at low frequency after vascular injury and that only a subset of expanding clones migrate, which together drives formation of oligoclonal neointimal lesions. VSMC contribution in small atherosclerotic lesions is typically from one or two clones, similar to observations in mature lesions. Low frequency (<0.1%) of clonal VSMC proliferation is also observed in vitro. Single-cell RNA-sequencing revealed progressive cell state changes across a contiguous VSMC population at onset of injury-induced proliferation. Proliferating VSMCs mapped selectively to one of two distinct trajectories and were associated with cells showing extensive phenotypic switching. A proliferation-associated transitory state shared pronounced similarities with atypical SCA1+ VSMCs from uninjured mouse arteries and VSMCs in healthy human aorta. We show functionally that clonal expansion of SCA1+ VSMCs from healthy arteries occurs at higher rate and frequency compared with SCA1- cells.ConclusionOur data suggest that activation of proliferation at low frequency is a general, cell-intrinsic feature of VSMCs. We show that rare VSMCs in healthy arteries display VSMC phenotypic switching akin to that observed in pathological vessel remodelling and that this is a conserved feature of mouse and human healthy arteries. The increased proliferation of modulated VSMCs from healthy arteries suggests that these cells respond more readily to disease-inducing cues and could drive oligoclonal VSMC expansion.

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Impedimetric Detection and Electromediated Apoptosis of Vascular Smooth Muscle Using Microfabricated Biosensors for Diagnosis and Therapeutic Intervention in Cardiovascular Diseases

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