Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Harhun, Maksym I.; Huggins, Christopher; Ratnasingham, Kumaran; Raje, Durgesh; Moss, R.E.; Szewczyk, Kinga; Vasilikostas, Georgios; Greenwood, Iain A.; Khong, Teck K; Wan, Andrew; Reddy, Marcus

doi:10.1111/j.1582-4934.2012.01609.x

Cited by 8 publications

(7 citation statements)

References 64 publications

(95 reference statements)

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“…Fluorescent immunodetection of proteins using specific antibodies to P2X1 and P2X4 receptors at 1:300 dilution was performed according to protocols previously described [10]. Fluorescence was visualised using high resolution x-y mode of a Zeiss LSM 510 laser scanning confocal microscope.…”

Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

“…Adipose tissue containing branches of human gastro-omental arteries (HGOAs) was dissected during surgery, and immediately transferred into cold (4°C) physiological saline solution (PSS) following composition of (mmol/l): KCl 6, NaCl 120, MgCl 2 1.2, CaCl 2 2.0, D-glucose 10 and HEPES 10; pH adjusted to 7.4 with NaOH. Blood vessels were then dissected as described previously [10]. The arteries were cleaned of connective tissue ( Supplementary Fig.…”

Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

“…Gene and protein expression analysis Total RNA extraction, reverse transcription reaction and PCR were performed as in our previous experiments [10]. Thirty-five amplification cycles were performed for samples from whole arterial fragments, and 45 amplification cycles were performed for cDNA samples from individually collected cells.…”

Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

“…The contractile properties of isolated myocytes were confirmed with application of high potassium (60 mmol/l) solution to the experimental chamber. The non-contractile cells displaying morphological properties of the phenotypically modulated vascular myocytes, such as filopodia, [10,12,13] were omitted during collection. The description of the cell collection procedure can be found in our previous work [10].…”

Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

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Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Nichols

Povstyan

Albert

et al. 2014

Purinergic Signalling

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Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca 2+ ] i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca 2+ ] i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

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Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

Section: Tissue Retrieval and Preparationmentioning

confidence: 99%

See 2 more Smart Citations

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Nichols

Povstyan

Albert

et al. 2014

Purinergic Signalling

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“…20 Reverse transcription polymerase chain reaction gene expression analysis and immunocytochemical detection of proteins and data analysis were performed as previously described. 21 See the online-only Data Supplement for the Western blotting protocols, lists of primers, and antibodies used.…”

Section: Gene and Protein Expression Calcium Imaging And Electrophymentioning

confidence: 99%

ATP-Evoked Sustained Vasoconstrictions Mediated by Heteromeric P2X1/4 Receptors in Cerebral Arteries

Harhun

Povstyan

Albert

et al. 2014

Stroke

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Background and Purpose-Current knowledge states that vasoconstrictor responses to ATP are mediated by rapidly desensitizing ligand-gated P2X1 receptors in vascular smooth muscle cells (VSMCs). However, ATP is implicated in contributing to pathological conditions involving sustained vasoconstrictor response such as cerebral vasospasm. The purpose of this study is to test the hypothesis that the stimulation of VSMC P2XR receptors (P2XRs) contributes to ATPevoked sustained vasoconstrictions in rat middle cerebral arteries (RMCAs). Methods-Reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used to analyze expression of mRNA and proteins in RMCAs VSMCs. Ionic currents and calcium responses were investigated using patch-clamp and confocal imaging techniques, respectively. Functional responses were confirmed using wire myography. Results-Expression of mRNA and protein for P2X1R and P2X4R subunits was identified in RMCA VSMCs. Confocal imaging in fluo-3-loaded VSMCs showed that ATP and a selective P2XR agonist, αβmeATP, evoked similar dosedependent increases in [Ca 2+ ] i . Patch-clamp experiments identified 2 components of P2XR-mediated currents: consisting of a fast desensitizing phase mediated by homomeric P2X1Rs and a slowly desensitizing phase involving heteromeric P2X1/4Rs. Isometric tension measurements showed that ≈80%:20% of initial ATP-evoked vasoconstriction in RMCA is mediated by homomeric P2X1Rs and heteromeric P2X1/4Rs, respectively. The sustained slowly desensitizing and rapidly recovering from desensitization responses are mediated by heteromeric P2X1/4Rs. Conclusions-This study reveals for the first time that apart from rapidly desensitizing homomeric P2X1Rs, heteromeric P2X1/4Rs contribute to the sustained component of the purinergic-mediated vasoconstriction in RMCA. Our study, therefore, identifies possible novel targets for therapeutical intervention in cerebral circulation.

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Characterization of transcriptional and posttranscriptional properties of native and cultured phenotypically modulated vascular smooth muscle cells

2012

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Various in vitro models are used for studying phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of vascular smooth muscle cells (cVSMCs) is most often used for this purpose. On the other hand, vascular interstitial cells (VICs) are native phenotypically modulated VSMCs present in blood vessels under normal physiological conditions. The aim of this work has been to compare the difference in expression of a number of VSMC-specific markers, which are commonly used for the characterisation of phenotypic modulation of VSMCs, between freshly dispersed VSMCs, VICs and cVSMCs from rat abdominal aorta. Our experiments show that VICs are present in the rat aorta and express markers of VSMCs. Both VICs and cVSMCs display the presence of sparse individual stress fibres enriched in alpha smooth muscle actin (αSM-actin), whereas in VSMCs, this protein is more densely packed. Compared with contractile VSMCs, both VICs and cVSMCs display decreased expression of VSMC-specific markers such as smoothelin, myosin light chain kinase and SM22α; however, the expression of two major cytoskeletal and contractile proteins (smooth muscle myosin heavy chain and αSM-actin) was downregulated in cVSMCs but not in VICs compared with contractile VSMCs. These results suggest different mechanisms for the phenotypic modulation of cVSMCs and VICs. VICs might therefore represent a novel convenient model for studying molecular mechanisms that govern the phenotypic modulation of VSMCs.

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Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Cited by 8 publications

References 64 publications

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

ATP-Evoked Sustained Vasoconstrictions Mediated by Heteromeric P2X1/4 Receptors in Cerebral Arteries

Characterization of transcriptional and posttranscriptional properties of native and cultured phenotypically modulated vascular smooth muscle cells

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