Dataset Information

Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart.

ABSTRACT: The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in the heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. We developed an integrated process for simultaneous isolation and culture of the main cell types comprising the microcirculation in adult mouse heart: endothelial cells, pericytes, and vascular smooth muscle cells. These cell types were characterized with isobaric labeling quantitative proteomics and mRNA sequencing. We defined microvascular cell proteomes, identified novel protein markers, and confirmed established cell-specific markers. Our results allow identification of unique markers and regulatory proteins that govern microvascular physiology and pathology.

SUBMITTER: Cao Z

PROVIDER: S-EPMC9132906 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart.

Cao Zhiping Z Minnier Jessica J Liu Lijuan L Scott Kristin L Lyon KLL Reddy Ashok P AP Wilmarth Phillip A PA David Larry L LL Barnes Anthony P AP Grafe Marjorie R MR Kaul Sanjiv S Alkayed Nabil J NJ Davis Catherine M CM

Scientific reports 20220525 1

The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of micr ...[more]

PMID: 35614104

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Project description:The microcirculation serves crucial functions in adult heart that are distinct from those carried out by epicardial vessels. Microvessels are also governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. While great progress has been made in understanding and treating coronary artery disease (CAD), there are few treatment options for patients with microvascular heart disease, primarily due to our limited understanding of underlying pathology. The advent of high-throughput analytical approaches of mRNA and protein expression in specific cells provides an opportunity to transform our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in an adult heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. Furthermore, in-vitro functional and molecular analysis requires an integrated workflow that combines primary cell culture with high-throughput proteomic or transcriptomic analysis. Our goal was an integrated process for simultaneous isolation, culture, and proteomic profiling of the three main cell types comprising the microcirculation in adult mouse heart: endothelial cells (ECs), pericytes (PCs) and vascular smooth muscle cells (VSMCs). We developed an integrated platform for simultaneous isolation and culture of ECs, PCs and VSMCs from adult mouse heart, coupled with unbiased mass spectrometry (MS)-based characterization of protein expression in these cells. We defined microvascular cell proteomes, identified novel protein markers and confirmed established cell-specific markers. Isolating and analyzing microvascular cell types separately from the same preparation of adult mouse hearts allowed for separate investigations into the unique contributions of the different cell types to health and disease, and interactions among different cell types.

Project description:Many pathological conditions of the brain are associated with structural abnormalities within the neurovascular system and linked to pericyte (PC) loss and/or dysfunction. Since crosstalk between endothelial cells (ECs) and PCs greatly impacts the function of the blood-brain barrier (BBB), effects of PCs on endothelial integrity and function have been investigated extensively. However, the impact of ECs on the function and activity of PCs remains largely unknown. Hence, using co-cultures of human brain vascular PCs with human cerebral microvascular ECs on opposite sides of porous Transwell inserts which facilitates direct EC-PC contact and improves EC barrier function, we analyzed EC-driven transcriptomic changes in PCs using microarrays and changes in cytokines/chemokines using proteome arrays. Gene expression analysis (GEA) in PCs co-cultured with ECs versus PCs cultured alone showed significant upregulation of 1'334 genes and downregulation of 964 genes. GEA in co-cultured PCs revealed increased expression of five prominent PC markers as well as soluble factors, such as transforming growth factor beta, fibroblast growth factor, angiopoietin 1, brain-derived neurotrophic factor, all of which are involved in EC-PC crosstalk and BBB induction. Pathway enrichment analysis of modulated genes showed a strong impact on many inflammatory and extracellular matrix (ECM) pathways including interferon and interleukin signaling, TGF-β and interleukin-1 regulation of ECM, as well as on the mRNA processing pathway. Interestingly, while co-culture induced the mRNA expression of many chemokines and cytokines, including several CCL- and CXC-motif ligands and interleukins, we observed a decreased expression of the same inflammatory mediators on the protein level. Importantly, in PCs, ECs significantly induced interferon associated proteins (IFIT1, IFI44L, IF127, IFIT3, IFI6, IFI44) with anti-viral actions; downregulated prostaglandin E receptor 2 (prevent COX-2 mediated BBB damage); upregulated fibulin-3 and connective tissue growth factor essential for BBB integrity; and multiple ECMs (collagens and integrins) that inhibit cell migration. Our findings suggest that via direct contact, ECs prime PCs to induce molecules to promote BBB integrity and cell survival during infection and inflammatory insult. Taken together, we provide first evidence that interaction with ECs though porous membranes induces major changes in the transcriptomic and proteomic profile of PCs. ECs influence genes involved in diverse aspects of PC function including PC maturation, cell survival, anti-viral defense, blood flow regulation, immuno-modulation and ECM deposition.

Project description:Pericytes are unique, multi-functional mural cells localized at the abluminal side of the perivascular space in microvessels. Originally discovered in 19th century, pericytes had drawn less attention until decades ago mainly due to lack of specific markers. Recently, however, a growing body of evidence has revealed that pericytes play various important roles: development and maintenance of blood-brain barrier (BBB), regulation of the neurovascular system (e.g., vascular stability, vessel formation, cerebral blood flow, etc.), trafficking of inflammatory cells, clearance of toxic waste products from the brain, and acquisition of stem cell-like properties. In the neurovascular unit, pericytes perform these functions through coordinated crosstalk with neighboring cells including endothelial, glial, and neuronal cells. Dysfunction of pericytes contribute to a wide variety of diseases that lead to cognitive impairments such as cerebral small vessel disease (SVD), acute stroke, Alzheimer's disease (AD), and other neurological disorders. For instance, in SVDs, pericyte degeneration leads to microvessel instability and demyelination while in stroke, pericyte constriction after ischemia causes a no-reflow phenomenon in brain capillaries. In AD, which shares some common risk factors with vascular dementia, reduction in pericyte coverage and subsequent microvascular impairments are observed in association with white matter attenuation and contribute to impaired cognition. Pericyte loss causes BBB-breakdown, which stagnates amyloid ? clearance and the leakage of neurotoxic molecules into the brain parenchyma. In this review, we first summarize the characteristics of brain microvessel pericytes, and their roles in the central nervous system. Then, we focus on how dysfunctional pericytes contribute to the pathogenesis of vascular cognitive impairment including cerebral 'small vessel' and 'large vessel' diseases, as well as AD. Finally, we discuss therapeutic implications for these disorders by targeting pericytes.

Dataset Information

Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart.

Publications

Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart.

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