Pía C. Burboa, Juliana C. Corrêa-Velloso, Cecilia Arriagada, Andrew P. Thomas, Walter N. Durán, Mauricio A. Lillo
{"title":"基质凝胶变化对微血管内皮细胞原代培养功能的影响","authors":"Pía C. Burboa, Juliana C. Corrêa-Velloso, Cecilia Arriagada, Andrew P. Thomas, Walter N. Durán, Mauricio A. Lillo","doi":"10.1111/micc.12859","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Objective</h3>\n \n <p>The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident.</p>\n \n <p>Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca<sup>2+</sup> elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.</p>\n </section>\n </div>","PeriodicalId":18459,"journal":{"name":"Microcirculation","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11227414/pdf/","citationCount":"0","resultStr":"{\"title\":\"Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function\",\"authors\":\"Pía C. Burboa, Juliana C. Corrêa-Velloso, Cecilia Arriagada, Andrew P. Thomas, Walter N. Durán, Mauricio A. Lillo\",\"doi\":\"10.1111/micc.12859\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Objective</h3>\\n \\n <p>The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident.</p>\\n \\n <p>Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca<sup>2+</sup> elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. 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Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function
Objective
The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques.
Methods
Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology.
Results
Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident.
Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca2+ elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs.
Conclusions
Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.
期刊介绍:
The journal features original contributions that are the result of investigations contributing significant new information relating to the vascular and lymphatic microcirculation addressed at the intact animal, organ, cellular, or molecular level. Papers describe applications of the methods of physiology, biophysics, bioengineering, genetics, cell biology, biochemistry, and molecular biology to problems in microcirculation.
Microcirculation also publishes state-of-the-art reviews that address frontier areas or new advances in technology in the fields of microcirculatory disease and function. Specific areas of interest include: Angiogenesis, growth and remodeling; Transport and exchange of gasses and solutes; Rheology and biorheology; Endothelial cell biology and metabolism; Interactions between endothelium, smooth muscle, parenchymal cells, leukocytes and platelets; Regulation of vasomotor tone; and Microvascular structures, imaging and morphometry. Papers also describe innovations in experimental techniques and instrumentation for studying all aspects of microcirculatory structure and function.