Alterations in polypeptide and gene expression during In vitro cytodifferentiation of smooth muscle cells

Juxtaglomerular (JG) cells, major sources of renin, differentiate from metanephric mesenchymal cells that give rise to JG cells or a subset of smooth muscle cells of the renal afferent arteriole. During periods of dehydration and salt deprivation, renal mesenchymal stromal cells (MSCs) differentiate from JG cells. JG cells undergo expansion and smooth muscle cells redifferentiate to express renin along the afferent arteriole. Gene expression profiling comparing resident renal MSCs with JG cells indicates that the transcription factor Sox6 is highly expressed in JG cells in the adult kidney. In vitro, loss of Sox6 expression reduces differentiation of renal MSCs to renin-producing cells. In vivo, Sox6 expression is upregulated after a low-Na+ diet and furosemide. Importantly, knockout of Sox6 in Ren1d+ cells halts the increase in renin-expressing cells normally seen during a low-Na+ diet and furosemide as well as the typical increase in renin. Furthermore, Sox6 ablation in renin-expressing cells halts the recruitment of smooth muscle cells along the afferent arteriole, which normally express renin under these conditions. These results support a previously undefined role for Sox6 in renin expression.

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ECM gene expression correlates with in vitro tissue growth and development in fibrin gel remodeled by neonatal smooth muscle cells

Matrix Biology ◽

10.1016/s0945-053x(03)00078-7 ◽

2003 ◽

Vol 22 (6) ◽

pp. 477-490 ◽

Cited By ~ 98

Author(s):

J.J Ross ◽

R.T Tranquillo

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Growth And Development ◽

Muscle Cells ◽

Tissue Growth ◽

Fibrin Gel

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243. In vitro culture significantly reduces differences in gene expression profiles between myometrial and fibroid smooth muscle cells

Reproduction Fertility and Development ◽

10.1071/srb05abs243 ◽

2005 ◽

Vol 17 (9) ◽

pp. 96

Author(s):

M. Zaitseva ◽

P. A. W. Rogers

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cultured Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Muscle Cells ◽

Compare Gene Expression

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMC) have been widely used as a model for the study of fibroid growth. Although it has been shown that FSMC can behave differently in culture to MSMC, it is not clear how relevant the cultured cells and their responses are to the in-vivo situation. The aim of the present study was to compare gene expression profiles of M and F tissue to cells isolated from the same tissue and cultured for up to 3 passages. M and F were collected from hysterectomy specimens (n = 6), part was snap frozen for RNA and the rest used to isolate SMC, which were cultured for 3 passages and RNA was collected at passage 0 (P0) and 3 (P3). 36 microarrays were performed on 8K human cDNA slides, 6 per each specimen (3 for M and 3 for F: tissue, cell at P0 and P3) against reference RNA. Analysis revealed significant differences between tissues and cultured cells. Independent clustering assigned tissues versus cells into two distinct groups based on their expression profiles. Parametric ANOVA with Benjamini-Hochberg correction and post-hoc testing was used to determine similarities and differences between tissues and cells. 128 genes were found to be statistically different between M and F tissue, 66 between MSMC and FSMC at P0, and only 9 at P3. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Similar numbers of genes were up regulated as were down regulated. Expression profiles of genes of interest including estrogen receptor α and progesterone receptor were also validated using real-time PCR. This is the first study to compare gene expression of in vivo and in vitro fibroid and myometrial SMC. The results demonstrate that large changes occur in SMC gene expression in culture, reducing differences between myometrial and fibroid cells. This study indicates that results of in vitro studies should be interpreted with caution as many genes have an altered gene expression profile in culture.

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In vitro culture significantly alters gene expression profiles and reduces differences between myometrial and fibroid smooth muscle cells

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gal018 ◽

2006 ◽

Vol 12 (3) ◽

pp. 187-207 ◽

Cited By ~ 59

Author(s):

Marina Zaitseva ◽

Beverley J. Vollenhoven ◽

Peter A.W. Rogers

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

In Vitro Culture ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Muscle Cells

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Deficiency in Aim2 affects viability and calcification of vascular smooth muscle cells from murine aortas and Angiotensin-II induced aortic aneurysms

10.21203/rs.3.rs-29275/v2 ◽

2020 ◽

Author(s):

Markus Wortmann ◽

Muhammad Arshad ◽

Maani Hakimi ◽

Dittmar Böckler ◽

Susanne Dihlmann

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Angiotensin Ii ◽

Aortic Aneurysm ◽

Mouse Model ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells

Abstract Background: Phenotypic transformation of vascular smooth muscle cells is a key element in vascular remodeling and aortic aneurysm growth. Previously, deletion of several inflammasome components decreased formation of aortic aneurysm (AA) in the Angiotensin II (AngII) -induced mouse model. We hypothesized that the inflammasome sensor Absent in melanoma 2 (Aim2) might affect the phenotype of vascular smooth muscle cells (VSMC), thereby reducing AA formation. Methods : Aim2-/- mice and wild-type (WT) C57Bl/6J mice were used as an animal model. VSMC were isolated from 6 months old mice and grown in vitro . Young (passage 3-5) and senescent (passage 7-12) cells were analyzed in vitro for calcification in mineralization medium by Alizarin Red S staining. Expression of calcification and inflammatory markers were studied by real-time RT-PCR and Western blotting, release of cytokines was determined by ELISA. To induce AA, osmotic mini-pumps loaded with AngII (1500 ng/kg bodyweight/min) were implanted for 28 days in male mice at 6 months of age. Results : Compared with VSMC from WT mice, VSMC isolated from Aim2-/- mice were larger, less viable, and underwent stronger calcification in mineralization medium, along with induction of Bmp4 and repression of Tnfsf11/Rankl gene expression. In addition, Aim2 deficiency was associated with reduced inflammasome gene expression and release of Interleukin-6. Using the mouse model of AngII induced AA, Aim2 deficiency reduced AA incidence to 48.4% (15/31) in Aim2-/- mice versus 76.5% (13/17) in WT mice. In contrast to Aim2-/- mice, AA from WT mice expressed significantly increased levels of alpha-smooth muscle actin/ Acta2 , indicating tissue remodeling. Reduced cell proliferation in Aim2-/- mice was indicated by significantly increased p16ink4a/ Cdkn2a expression in untreated and AngII-infused aortas, and by significantly lower amounts of proliferating (Ki67 positive) VSMC in AngII-infused Aim2-/- mice. Conclusions: Our results suggest a role for Aim2 in regulating VSMC proliferation and transition to an osteoblast-like or osteoclast-like phenotype, thereby modulating the response of VSMC in aortic remodeling and AA Formation.

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Deficiency in Aim2 affects viability and calcification of vascular smooth muscle cells from murine aortas and Angiotensin-II induced aortic aneurysms

10.21203/rs.3.rs-29275/v3 ◽

2020 ◽

Author(s):

Markus Wortmann ◽

Muhammad Arshad ◽

Maani Hakimi ◽

Dittmar Böckler ◽

Susanne Dihlmann

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Angiotensin Ii ◽

Aortic Aneurysm ◽

Mouse Model ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells

Abstract Background: Phenotypic transformation of vascular smooth muscle cells is a key element in vascular remodeling and aortic aneurysm growth. Previously, deletion of several inflammasome components decreased formation of aortic aneurysm (AA) in the Angiotensin II (AngII) -induced mouse model. We hypothesized that the inflammasome sensor Absent in melanoma 2 (Aim2) might affect the phenotype of vascular smooth muscle cells (VSMC), thereby reducing AA formation. Methods: Aim2-/- mice and wild-type (WT) C57Bl/6J mice were used as an animal model. VSMC were isolated from 6 months old mice and grown in vitro. Young (passage 3-5) and senescent (passage 7-12) cells were analyzed in vitro for calcification in mineralization medium by Alizarin Red S staining. Expression of calcification and inflammatory markers were studied by real-time RT-PCR and Western blotting, release of cytokines was determined by ELISA. To induce AA, osmotic mini-pumps loaded with AngII (1500 ng/kg bodyweight/min) were implanted for 28 days in male mice at 6 months of age.Results: Compared with VSMC from WT mice, VSMC isolated from Aim2-/- mice were larger, less viable, and underwent stronger calcification in mineralization medium, along with induction of Bmp4 and repression of Tnfsf11/Rankl gene expression. In addition, Aim2 deficiency was associated with reduced inflammasome gene expression and release of Interleukin-6. Using the mouse model of AngII induced AA, Aim2 deficiency reduced AA incidence to 48.4% (15/31) in Aim2-/- mice versus 76.5% (13/17) in WT mice. In contrast to Aim2-/- mice, AA from WT mice expressed significantly increased levels of alpha-smooth muscle actin/Acta2, indicating tissue remodeling. Reduced cell proliferation in Aim2-/- mice was indicated by significantly increased p16ink4a/Cdkn2a expression in untreated and AngII-infused aortas, and by significantly lower amounts of proliferating (Ki67 positive) VSMC in AngII-infused Aim2-/- mice. Conclusions: Our results suggest a role for Aim2 in regulating VSMC proliferation and transition to an osteoblast-like or osteoclast-like phenotype, thereby modulating the response of VSMC in aortic remodeling and AA formation.

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Deficiency in Aim2 affects viability and calcification of vascular smooth muscle cells from murine aortas and angiotensin-II induced aortic aneurysms

Molecular Medicine ◽

10.1186/s10020-020-00212-z ◽

2020 ◽

Vol 26 (1) ◽

Author(s):

Markus Wortmann ◽

Muhammad Arshad ◽

Maani Hakimi ◽

Dittmar Böckler ◽

Susanne Dihlmann

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Angiotensin Ii ◽

Aortic Aneurysm ◽

Mouse Model ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells

Abstract Background Phenotypic transformation of vascular smooth muscle cells is a key element in vascular remodeling and aortic aneurysm growth. Previously, deletion of several inflammasome components decreased formation of aortic aneurysm (AA) in the Angiotensin II (AngII) -induced mouse model. We hypothesized that the inflammasome sensor Absent in melanoma 2 (Aim2) might affect the phenotype of vascular smooth muscle cells (VSMC), thereby reducing AA formation. Methods Aim2−/− mice and wild-type (WT) C57Bl/6 J mice were used as an animal model. VSMC were isolated from 6 months old mice and grown in vitro. Young (passage 3–5) and senescent (passage 7–12) cells were analyzed in vitro for calcification in mineralization medium by Alizarin Red S staining. Expression of calcification and inflammatory markers were studied by real-time RT-PCR and Western blotting, release of cytokines was determined by ELISA. To induce AA, osmotic mini-pumps loaded with AngII (1500 ng/kg bodyweight/min) were implanted for 28 days in male mice at 6 months of age. Results Compared with VSMC from WT mice, VSMC isolated from Aim2−/− mice were larger, less viable, and underwent stronger calcification in mineralization medium, along with induction of Bmp4 and repression of Tnfsf11/Rankl gene expression. In addition, Aim2 deficiency was associated with reduced inflammasome gene expression and release of Interleukin-6. Using the mouse model of AngII induced AA, Aim2 deficiency reduced AA incidence to 48.4% (15/31) in Aim2−/− mice versus 76.5% (13/17) in WT mice. In contrast to Aim2−/− mice, AA from WT mice expressed significantly increased levels of alpha-smooth muscle actin/Acta2, indicating tissue remodeling. Reduced cell proliferation in Aim2−/− mice was indicated by significantly increased p16ink4a/Cdkn2a expression in untreated and AngII-infused aortas, and by significantly lower amounts of proliferating (Ki67 positive) VSMC in AngII-infused Aim2−/− mice. Conclusions Our results suggest a role for Aim2 in regulating VSMC proliferation and transition to an osteoblast-like or osteoclast-like phenotype, thereby modulating the response of VSMC in aortic remodeling and AA formation.

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