Smooth muscle αv integrins regulate vascular fibrosis via CD109 downregulation of TGF-ß signalling.

Aims: αv integrins are implicated in fibrosis in a number of organs through their ability to activate TGF-ß. However their role in vascular fibrosis and collagen accumulation is only partially understood. Here we have used αv conditional knockout mice and cell lines to determine how αv contributes to vascular smooth muscle cell (VSMC) function in vascular fibrosis and the role of TGF-ß in that process. Methods and results: Angiotensin II (Ang II) treatment causes upregulation of αv and ß3 expression in the vessel wall, associated with increased collagen deposition. We found that deletion of αv integrin subunit from VSMCs (αv SMKO) protected mice against angiotensin II-induced collagen production and assembly. Transcriptomic analysis of the vessel wall in αv SMKO mice and controls identified a significant reduction in expression of fibrosis and related genes in αv SMKO mice. In contrast, αv SMKO mice showed prolonged expression of CD109, which is known to affect TGF-ß signalling. Using cultured mouse and human VSMCs, we showed that overexpression of CD109 phenocopied knockdown of αv integrin, attenuating collagen expression, TGF-ß activation, and Smad2/3 signalling in response to angiotensin II or TGF-ß stimulation. CD109 and TGF-ß receptor were internalized in early endosomes. Conclusion: We identify a role for VSMC αv integrin in vascular fibrosis and show that αv acts in concert with CD109 to regulate TGF-ß signalling.

Absence of Desmin Results in Impaired Adaptive Response to Mechanical Overloading of Skeletal Muscle.

Background: Desmin is a muscle-specific protein belonging to the intermediate filament family. Desmin mutations are linked to skeletal muscle defects, including inherited myopathies with severe clinical manifestations. The aim of this study was to examine the role of desmin in skeletal muscle remodeling and performance gain induced by muscle mechanical overloading which mimics resistance training. Methods: Plantaris muscles were overloaded by surgical ablation of gastrocnemius and soleus muscles. The functional response of plantaris muscle to mechanical overloading in desmin-deficient mice (DesKO, n = 32) was compared to that of control mice (n = 36) after 7-days or 1-month overloading. To elucidate the molecular mechanisms implicated in the observed partial adaptive response of DesKO muscle, we examined the expression levels of genes involved in muscle growth, myogenesis, inflammation and oxidative energetic metabolism. Moreover, ultrastructure and the proteolysis pathway were explored. Results: Contrary to control, absolute maximal force did not increase in DesKO muscle following 1-month mechanical overloading. Fatigue resistance was also less increased in DesKO as compared to control muscle. Despite impaired functional adaptive response of DesKO mice to mechanical overloading, muscle weight and the number of oxidative MHC2a-positive fibers per cross-section similarly increased in both genotypes after 1-month overloading. However, mechanical overloading-elicited remodeling failed to activate a normal myogenic program after 7-days overloading, resulting in proportionally reduced activation and differentiation of muscle stem cells. Ultrastructural analysis of the plantaris muscle after 1-month overloading revealed muscle fiber damage in DesKO, as indicated by the loss of sarcomere integrity and mitochondrial abnormalities. Moreover, the observed accumulation of autophagosomes and lysosomes in DesKO muscle fibers could indicate a blockage of autophagy. To address this issue, two main proteolysis pathways, the ubiquitin-proteasome system and autophagy, were explored in DesKO and control muscle. Our results suggested an alteration of proteolysis pathways in DesKO muscle in response to mechanical overloading. Conclusion: Taken together, our results show that mechanical overloading increases the negative impact of the lack of desmin on myofibril organization and mitochondria. Furthermore, our results suggest that under these conditions, the repairing activity of autophagy is disturbed. Consequently, force generation is not improved despite muscle growth, suggesting that desmin is required for a complete response to resistance training in skeletal muscle.

Effects of the selective inhibition of proteasome caspase-like activity by CLi a derivative of nor-cerpegin in dystrophic mdx mice.

Previous studies have shown that proteasome inhibition can have beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of a new selective proteasome inhibitor, CLi, a strong caspase-like inhibitor of the 20S proteasome, on skeletal and cardiac muscle functions of mdx mice. In the first series of experiments, five-month-old male mdx mice (n = 34) were treated with 2 different doses (20 and 100 µg/kg) of CLi and in the second series of experiments, five-month-old female mdx (n = 19) and wild-type (n = 24) mice were treated with 20 µg/kg CLi and Velcade (1 mg/kg) for 1-month. All animals were treadmill exercised twice a week to worsen the dystrophic features. In the first series of experiments, our results demonstrated that 20 µg/kg CLi did not significantly increase absolute and specific maximal forces in skeletal muscle from male mdx mice. Moreover, the higher susceptibility to contraction induced skeletal muscle injury was worsened by 100 µg/kg CLi since the force drop following lengthening contractions was increased with this high dose. Furthermore, we found no differences in the mRNA levels of the molecular markers implicated in dystrophic features. Concerning cardiac function, CLi had no effect on left ventricular function since ejection and shortening fractions were unchanged in male mdx mice. Similarly, CLi did not modify the expression of genes implicated in cardiac remodeling. In the second series of experiments, our results demonstrated an improvement in absolute and specific maximal forces by CLi, whereas Velcade only increased specific maximal force in female mdx mice. In addition, exercise tolerance was not improved by CLi. Taken together, our results show that CLi treatment can only improve maximal force production in exercised female mdx mice without affecting either exercice tolerance capacity or cardiac function. In conclusion, selective inhibition of caspase-like activity of proteasome with CLi has no compelling beneficial effect in dystrophic mdx mice.

Publisher Correction: Vimentin knockout results in increased expression of sub-endothelial basement membrane components and carotid stiffness in mice.

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Vimentin knockout results in increased expression of sub-endothelial basement membrane components and carotid stiffness in mice.

Intermediate filaments are involved in stress-related cell mechanical properties and in plasticity via the regulation of focal adhesions (FAs) and the actomyosin network. We investigated whether vimentin regulates endothelial cells (ECs) and vascular smooth muscle cells (SMCs) and thereby influences vasomotor tone and arterial stiffness. Vimentin knockout mice (Vim-/-) exhibited increased expression of laminin, fibronectin, perlecan, collagen IV and VE-cadherin as well as von Willebrand factor deposition in the subendothelial basement membrane. Smooth muscle (SM) myosin heavy chain, α-SM actin and smoothelin were decreased in Vim-/- mice. Electron microscopy revealed a denser endothelial basement membrane and increased SM cell-matrix interactions. Integrin αv, talin and vinculin present in FAs were increased in Vim-/- mice. Phosphorylated FA kinase and its targets Src and ERK1/2 were elevated in Vim-/- mice. Knockout of vimentin, but not of synemin, resulted in increased carotid stiffness and contractility and endothelial dysfunction, independently of blood pressure and the collagen/elastin ratio. The increase in arterial stiffness in Vim-/- mice likely involves vasomotor tone and endothelial basement membrane organization changes. At the tissue level, the results show the implication of FAs both in ECs and vascular SMCs in the role of vimentin in arterial stiffening.

Synemin acts as a regulator of signalling molecules during skeletal muscle hypertrophy.

Synemin, a type IV intermediate filament (IF) protein, forms a bridge between IFs and cellular membranes. As an A-kinase-anchoring protein, it also provides temporal and spatial targeting of protein kinase A (PKA). However, little is known about its functional roles in either process. To better understand its functions in muscle tissue, we generated synemin-deficient (Synm(-) (/-)) mice. Synm(-) (/-) mice displayed normal development and fertility but showed a mild degeneration and regeneration phenotype in myofibres and defects in sarcolemma membranes. Following mechanical overload, Synm(-) (/-) mice muscles showed a higher hypertrophic capacity with increased maximal force and fatigue resistance compared with control mice. At the molecular level, increased remodelling capacity was accompanied by decreased myostatin (also known as GDF8) and atrogin (also known as FBXO32) expression, and increased follistatin expression. Furthermore, the activity of muscle-mass control molecules (the PKA RIIα subunit, p70S6K and CREB1) was increased in mutant mice. Finally, analysis of muscle satellite cell behaviour suggested that the absence of synemin could affect the balance between self-renewal and differentiation of these cells. Taken together, our results show that synemin is necessary to maintain membrane integrity and regulates signalling molecules during muscle hypertrophy.

SRF selectively controls tip cell invasive behavior in angiogenesis.

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Inactivation of serum response factor contributes to decrease vascular muscular tone and arterial stiffness in mice.

RATIONALE: Vascular smooth muscle (SM) cell phenotypic modulation plays an important role in arterial stiffening associated with aging. Serum response factor (SRF) is a major transcription factor regulating SM genes involved in maintenance of the contractile state of vascular SM cells. OBJECTIVE: We investigated whether SRF and its target genes regulate intrinsic SM tone and thereby arterial stiffness. METHODS AND RESULTS: The SRF gene was inactivated SM-specific knockout of SRF (SRF(SMKO)) specifically in vascular SM cells by injection of tamoxifen into adult transgenic mice. Fifteen days later, arterial pressure and carotid thickness were lower in SRF(SMKO) than in control mice. The carotid distensibility/pressure and elastic modulus/wall stress curves showed a greater arterial elasticity in SRF(SMKO) without modification in collagen/elastin ratio. In SRF(SMKO), vasodilation was decreased in aorta and carotid arteries, whereas a decrease in contractile response was found in mesenteric arteries. By contrast, in mice with inducible SRF overexpression, the in vitro contractile response was significantly increased in all arteries. Without endothelium, the contraction was reduced in SRF(SMKO) compared with control aortic rings owing to impairment of the NO pathway. Contractile components (SM-actin and myosin light chain), regulators of the contractile response (myosin light chain kinase, myosin phosphatase target subunit 1, and protein kinase C-potentiated myosin phosphatase inhibitor) and integrins were reduced in SRF(SMKO). CONCLUSIONS: SRF controls vasoconstriction in mesenteric arteries via vascular SM cell phenotypic modulation linked to changes in contractile protein gene expression. SRF-related decreases in vasomotor tone and cell-matrix attachment increase arterial elasticity in large arteries.

Laminin receptor involvement in the anti-angiogenic activity of pigment epithelium-derived factor.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A(2)/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44-77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120-210). A 25-mer peptide named P46 (aa 46-70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.

Serum response factor is required for sprouting angiogenesis and vascular integrity.

Serum response factor (SRF) is a transcription factor that controls the expression of cytoskeletal proteins and immediate early genes in different cell types. Here, we found that SRF expression is restricted to endothelial cells (ECs) of small vessels such as capillaries in the mouse embryo. EC-specific Srf deletion led to aneurysms and hemorrhages from 11.5 days of mouse development (E11.5) and lethality at E14.5. Mutant embryos presented a reduced capillary density and defects in EC migration, with fewer numbers of filopodia in tip cells and ECs showing defects in actin polymerization and intercellular junctions. We show that SRF is essential for the expression of VE-cadherin and beta-actin in ECs both in vivo and in vitro. Moreover, knockdown of SRF in ECs impaired VEGF- and FGF-induced in vitro angiogenesis. Taken together, our results demonstrate that SRF plays an important role in sprouting angiogenesis and small vessel integrity in the mouse embryo.

Role of alpha1beta1-integrin in arterial stiffness and angiotensin-induced arterial wall hypertrophy in mice.

We examined the arterial phenotype of mice lacking alpha(1)-integrin (alpha(1)(-/-)) at baseline and after 4 wk of ANG II or norepinephrine (NE) administration. Arterial mechanical properties were determined in the carotid artery (CA). Integrin expression, MAPK kinases, and focal adhesion kinase (FAK) were assessed in the aorta. No change in arterial pressure was observed in alpha(1)(-/-) mice. Elastic modulus-wall stress curves were similar in alpha(1)(-/-) and alpha(1)(+/+) animals, indicating no change in arterial stiffness. The rupture pressure was lower in alpha(1)(-/-) mice, demonstrating decreased mechanical strength. Lack of alpha(1)-integrin was accompanied by an increase in beta(1)-, alpha(v)-, and alpha(5)-integrins but no change in alpha(2)-integrin. ANG II increased medial cross-sectional area of the CA in alpha(1)(+/+), but not alpha(1)(-/-), mice, whereas equivalent pressor doses of NE did not produce a significant increase in either group. In alpha(1)(+/+) mice, ANG II induced alpha(1)-integrin expression and smooth muscle cell (SMC) hypertrophy in the CA in association with increased aortic expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain and phosphorylation of ERK1/2, p38 MAPK, and FAK. ANG II did not induce SMC hypertrophy or phosphorylation of p38 MAPK and FAK in alpha(1)(-/-) mice. A functional anti-alpha(1)-integrin antibody inhibited in vitro the ANG II-induced phosphorylation of FAK and p38 MAPK. In conclusion, alpha(1)(-/-) mice exhibit a reduced mechanical strength at baseline and a lack of ANG II-induced SMC hypertrophy. These results emphasize the importance of alpha(1)beta(1)-integrin in p38 MAPK and FAK phosphorylation during vascular hypertrophy in response to ANG II.