Endothelial cell-conditioned medium downregulates smooth muscle contractile protein expression

1997 ◽  
Vol 272 (2) ◽  
pp. C582-C591 ◽  
Author(s):  
S. M. Vernon ◽  
M. J. Campos ◽  
T. Haystead ◽  
M. M. Thompson ◽  
P. E. DiCorleto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Protein Expression ◽  
Conditioned Medium ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Contractile Protein ◽  
Alpha Actin ◽  
Cell Conditioned Medium

Smooth muscle cells (SMC) within atherosclerotic lesions proliferate and exhibit phenotypic modulation, but the contribution of vascular endothelium to this process is poorly understood. Our aim was to examine the effects of endothelial cell-conditioned medium (ECCM) on vascular SMC growth and differentiation. Rat aortic ECCM stimulated a ninefold increase in [3H]thymidine incorporation and downregulated smooth muscle-specific myosin heavy chain and alpha-actin synthesis in rat aortic SMC. These effects were not inhibited by antibodies to platelet-derived growth factor (PDGF)-BB or PDGF-AB or with a PDGF beta-receptor subunit. Treatment with PDGF-BB (at a concentration found in ECCM), PDGF-AA, basic fibroblast growth factor, endothelin-1, or transforming growth factor-beta did not reproduce these effects. The ECCM activities were sensitive to heat and trypsinization, were >30 kDa in molecular mass, and bound weakly to heparin-Sepharose. Our data indicate that cultured endothelial cells produce a factor(s) that downregulates contractile protein expression in SMC, which may contribute to SMC dedifferentiation and proliferation.

scholarly journals Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium.

1988 ◽  
Vol 106 (5) ◽  
pp. 1659-1665 ◽  
Author(s):  
R M Lyons ◽  
J Keski-Oja ◽  
H L Moses
Keyword(s):  
Growth Factor ◽  
Conditioned Medium ◽  
Transforming Growth Factor Beta ◽  
Acid Treatment ◽  
Transforming Growth Factor ◽  
Soft Agar ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Mild Acid ◽  
Cell Conditioned Medium

Transforming growth factor-beta (TGF beta) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGF beta were studied using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model. Active TGF beta was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGF beta was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGF beta as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. In an effort to define more physiological means of TGF beta activation, the effects of some proteases were tested. Plasmin and cathepsin D were found to generate 25-kD bands corresponding to the active form of TGF beta as shown by immunoprecipitation analysis of radiolabeled cell-conditioned medium. Plasmin treatment of the medium resulted in activity that was quantitatively similar to that of mild acid treatment as measured by radioreceptor and soft agar assays. In addition, the plasmin-generated activity was inhibited by anti-TGF beta antibodies. Sequential treatments of AKR-MCA cell-conditioned medium with mild acid followed by plasmin or plasmin followed by mild acid gave activation comparable to either treatment alone. The data suggest that conditioned medium may contain at least two different pools of latent TGF beta. One pool is resistant to mild acid and/or plasmin and requires strong acid or alkali treatment for activation. A second pool is activated by mild pH change and/or plasmin. Activation of this form of latent TGF beta may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGF beta-binding protein complex.

scholarly journals Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity.

1992 ◽  
Vol 118 (4) ◽  
pp. 901-909 ◽  
Author(s):  
R Flaumenhaft ◽  
M Abe ◽  
P Mignatti ◽  
D B Rifkin
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Conditioned Medium ◽  
Cell Cultures ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Endothelial Cell Cultures ◽  
Growth Factor Beta

Exposure of bovine aortic or capillary endothelial cells to basic FGF (bFGF) for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h. We hypothesized that the decrease in PA activity following bFGF stimulation was mediated by transforming growth factor beta (TGF-beta) formed from its inactive precursor. Conditioned medium collected from endothelial cells 36 h after a 1-h exposure to bFGF, but not control medium, inhibited basal levels of PA activity when transferred to confluent monolayers of bovine aortic endothelial cells. Antibody to TGF-beta neutralized the inhibitory activity of this conditioned medium, indicating that the medium contained active TGF-beta. Northern blot analysis and quantitation of acid activatable latent TGF-beta in conditioned medium demonstrated that bFGF exposure did not increase the amount of transcription or secretion of latent TGF-beta by the endothelial cells. Both aprotinin, an inhibitor of plasmin, and anti-urokinase type PA IgG blocked the generation of active TGF-beta in cultures exposed to bFGF. These results demonstrated that plasmin generated by uPA activity is required for the activation of latent TGF-beta in endothelial cell cultures treated with bFGF. Activation of TGF-beta by endothelial cells exposed to bFGF appears to limit both the degree and duration of PA stimulation. Thus, in bFGF-stimulated endothelial cell cultures, PA levels are controlled by a negative feedback loop: PA, whose expression is stimulated by bFGF, contributes to the formation of TGF-beta, which in turn opposes the effects of bFGF by limiting PA synthesis and activity. These studies suggest a role for TGF-beta in reversing the invasive stage of angiogenesis and contributing to the formation of quiescent capillaries.

scholarly journals Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells.

1988 ◽  
Vol 107 (1) ◽  
pp. 299-306 ◽  
Author(s):  
R S Blank ◽  
M M Thompson ◽  
G K Owens
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Smooth Muscle Cells ◽  
Transforming Growth Factor Beta ◽  
Cell Sorting ◽  
Transforming Growth Factor ◽  
Growth Factor Beta ◽  
Actin Expression ◽  
Alpha Actin

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.

scholarly journals Androgens Accentuate TGF‐β Dependent Erk/Smad Activation During Thoracic Aortic Aneurysm Formation in Marfan Syndrome Male Mice

2020 ◽  
Vol 9 (20) ◽  
Author(s):  
Yasushi Tashima ◽  
Hao He ◽  
Jason Z. Cui ◽  
Albert J. Pedroza ◽  
Ken Nakamura ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Smooth Muscle Cell ◽  
Marfan Syndrome ◽  
Transforming Growth Factor Beta ◽  
Aortic Root ◽  
Transforming Growth Factor ◽  
Aneurysm Growth ◽  
Growth Factor Beta

Background Male patients with Marfan syndrome have a higher risk of aortic events and root dilatation compared with females. The role androgens play during Marfan syndrome aneurysm development in males remains unknown. We hypothesized that androgens potentiate transforming growth factor beta induced Erk (extracellular‐signal‐regulated kinase)/Smad activation, contributing to aneurysm progression in males. Methods and Results Aortic diameters in Fbn1 C1039G/+ and littermate wild‐type controls were measured at ages 6, 8, 12, and 16 weeks. Fbn1 C1039G/+ males were treated with (1) flutamide (androgen receptor blocker) or (2) vehicle control from age 6 to 16 weeks and then euthanized. p‐Erk1/2, p‐Smad2, and matrix metalloproteinase (MMP) activity were measured in ascending/aortic root and descending aorta specimens. Fbn1 C1039G/+ male and female ascending/aortic root‐derived smooth muscle cells were utilized in vitro to measure Erk/Smad activation and MMP‐2 activity following dihydrotestosterone, flutamide or transforming growth factor beta 1 treatment. Fbn1 C1039G/+ males have increased aneurysm growth. p‐Erk1/2 and p‐Smad2 were elevated in ascending/aortic root specimens at age 16 weeks. Corresponding with enhanced Erk/Smad signaling, MMP‐2 activity was higher in Fbn1 C1039G/+ males. In vitro smooth muscle cell studies revealed that dihydrotestosterone potentiates transforming growth factor beta‐induced Erk/Smad activation and MMP‐2 activity, which is reversed by flutamide treatment. Finally, in vivo flutamide treatment reduced aneurysm growth via p‐Erk1/2 and p‐Smad2 reduction in Fbn1 C1039G/+ males. Conclusions Fbn1 C1039G/+ males have enhanced aneurysm growth compared with females associated with enhanced p‐Erk1/2 and p‐Smad2 activation. Mechanistically, in vitro smooth muscle cell studies suggested that dihydrotestosterone potentiates transforming growth factor beta induced Erk/Smad activation. As biological proof of concept, flutamide treatment attenuated aneurysm growth and p‐Erk1/2 and p‐Smad2 signaling in Fbn1 C1039G/+ males.

scholarly journals Reduced pericyte and tight junction coverage in old diabetic rats are associated with hyperglycemia-induced cerebrovascular pericyte dysfunction

2020 ◽  
Author(s):  
Yedan Liu ◽  
Huawei Zhang ◽  
Shaoxun Wang ◽  
Ya Guo ◽  
Xing Fang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Tight Junction ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Cognitive Impairments ◽  
Cerebral Hemodynamics ◽  
Alpha Smooth Muscle Actin ◽  
Atp Production

Diabetes mellitus (DM) is one of the primary pathological factors that contributes to aging-related cognitive impairments, but the underlying mechanisms remain unclear. We recently reported that old DM rats exhibited impaired myogenic responses of the cerebral arteries and arterioles, poor cerebral blood flow autoregulation, enhanced blood-brain barrier (BBB) leakage, and cognitive impairments. These changes were associated with diminished vascular smooth muscle cell contractile capability linked to elevated reactive oxygen species (ROS) and reduced ATP production. The present study, using a non-obese T2DN DM rat, isolated parenchymal arterioles (PAs), and cultured cerebral microvascular pericytes, examined whether cerebrovascular pericyte in DM is damaged and whether pericyte dysfunction may play a role in the regulation of cerebral hemodynamics and BBB integrity. We found that ROS and mitochondrial superoxide production were elevated in PAs isolated from old DM rats and in high glucose (HG)-treated alpha-smooth muscle actin positive pericytes. HG-treated pericytes displayed decreased contractile capability in association with diminished mitochondrial respiration and ATP production. Additionally, the expression of advanced glycation end products, transforming growth factor-beta, vascular endothelial growth factor, and fibronectin were enhanced, but claudin 5 and integrin β1 was reduced in the brain of old DM rats and HG-treated pericytes. Further, endothelial tight junction and pericyte coverage on microvessels were reduced in the cortex of old DM rats. These results demonstrate our previous findings that the impaired cerebral hemodynamics and BBB leakage and cognitive impairments in the same old DM model are associated with hyperglycemia-induced cerebrovascular pericyte dysfunction.

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