Thrombospondin-4 knockout in hypertension protects small-artery endothelial function but induces aortic aneurysms

Thrombospondin-4 (TSP-4) is a multidomain calcium-binding protein that has both intracellular and extracellular functions. As an extracellular matrix protein, it is involved in remodeling processes. Previous work showed that, in the cardiovascular system, TSP-4 expression is induced in the heart in response to experimental pressure overload and infarction injury. Intracellularly, it mediates the endoplasmic reticulum stress response in the heart. In this study, we explored the role of TSP-4 in hypertension. For this purpose, wild-type and TSP-4 knockout ( Thbs4 −/−) mice were treated with angiotensin II (ANG II). Hearts from ANG II-treated Thbs4 −/− mice showed an exaggerated hypertrophic response. Interestingly, aortas from Thbs4 −/− mice treated with ANG II showed a high incidence of aneurysms. In resistance arteries, ANG II-treated wild-type mice showed impaired endothelial-dependent relaxation. This was not observed in ANG II-treated Thbs4 −/− mice or in untreated controls. No differences were found in the passive pressure-diameter curves or stress-strain relationships, although ANG II-treated Thbs4 −/− mice showed a tendency to be less stiff, associated with thicker diameters of the collagen fibers as revealed by electron microscopy. We conclude that TSP-4 plays a role in hypertension, affecting cardiac hypertrophy, aortic aneurysm formation, as well as endothelial-dependent relaxation in resistance arteries.

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Abstract 15539: Single-cell Analysis of Aortic Tissues From Patients With Marfan Syndrome Reveals Changes in Smooth Muscle Cell Differentiation

Circulation ◽

10.1161/circ.142.suppl_3.15539 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Ashley Dawson ◽

Yanming Li ◽

Pingping Ren ◽

Hernan Vasquez ◽

Chen Zhang ◽

...

Keyword(s):

Smooth Muscle ◽

Single Cell ◽

Marfan Syndrome ◽

Immune Cells ◽

Matrix Protein ◽

Immune Cell ◽

Aortic Aneurysms ◽

Extracellular Matrix Protein ◽

Cardiac Transplant ◽

Control Tissue

Background: Thoracic aortic aneurysms associated with Marfan syndrome (MFS) carry a high risk of mortality; however, the molecular and cellular processes leading to aortopathy in this population remain poorly understood. We aimed to use single-cell RNA (scRNA) sequencing to define the non-immune cell populations present within the aortic wall in MFS, hypothesizing that these would differ from those of non-aneurysmal control tissue. Methods: We performed scRNA sequencing of ascending aortic aneurysm tissues from MFS patients (n=3) undergoing aneurysm repair and of age-matched, non-aneurysmal control tissue from cardiac transplant donors and recipients (n=4). The Seurat package in R was used for analysis. Differentially expressed genes were identified using edgeR. Results: Eighteen non-immune cell clusters were identified, with conserved gene expression of the largest of the clusters consistent with smooth muscle cells (SMCs; n=6), fibroblasts (n=3), and endothelial cells (n=3). The SMCs and fibroblasts exhibited graded changes in their expression of contractile and extracellular matrix protein genes, supportive of a phenotypic continuum. Additionally, we identified differences in the proportions of non-immune cells in MFS tissues compared to controls. In control tissues, the most common non-immune cells expressed markers of contractile SMC maturity including CNN1 , MYH11 , and SMTN . In contrast, the largest clusters in MFS tissue were most closely related to SMCs on correlation analysis, but displayed increased expression of cyclin genes as well as immune, endothelial, and fibroblast genes indicative of de-differentiated, proliferative SMCs. Additionally, expression of genes associated with SMC phenotypic maturity, including MYH11 and MYOCD , were significantly downregulated in several of the MFS SMC clusters. Conclusion: Our data demonstrate a phenotypic continuum between fibroblasts and SMCs, with aortas from patients with MFS exhibiting an increased proportion of de-differentiated, proliferative SMCs compared to controls. Additionally, markers of SMC maturity were downregulated in SMCs in MFS compared to controls. This may be due to disruption of signaling pathways that promote differentiation.

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Upregulation of α8β1-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-β1

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.5.c1457 ◽

2001 ◽

Vol 281 (5) ◽

pp. C1457-C1467 ◽

Cited By ~ 32

Author(s):

Gaétan Thibault ◽

Marie-Josée Lacombe ◽

Lynn M. Schnapp ◽

Alexandre Lacasse ◽

Fatiha Bouzeghrane ◽

...

Keyword(s):

Angiotensin Ii ◽

Growth Factor ◽

Matrix Protein ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Transforming Growth Factor Β1 ◽

Cardiac Fibroblast ◽

Extracellular Matrix Protein ◽

Ang Ii ◽

Tgf Β1

Using a novel pharmacological tool with125I-echistatin to detect integrins on the cell, we have observed that cardiac fibroblasts harbor five different RGD-binding integrins: α8β1, α3β1, α5β1, αvβ1, and αvβ3. Stimulation of cardiac fibroblasts by angiotensin II (ANG II) or transforming growth factor-β1 (TGF-β1) resulted in an increase of protein and heightening by 50% of the receptor density of α8β1-integrin. The effect of ANG II was blocked by an AT1, but not an AT2, receptor antagonist, or by an anti-TGF-β1 antibody. ANG II and TGF-β1 increased fibronectin secretion, smooth muscle α-actin synthesis, and formation of actin stress fibers and enhanced attachment of fibroblasts to a fibronectin matrix. The α8- and β1-subunits were colocalized by immunocytochemistry with vinculin or β3-integrin at focal adhesion sites. These results indicate that α8β1-integrin is an abundant integrin on rat cardiac fibroblasts. Its positive modulation by ANG II and TGF-β1 in a myofibroblast-like phenotype suggests the involvement of α8β1-integrin in extracellular matrix protein deposition and cardiac fibroblast adhesion.

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Thrombospondin 1 mediates renal dysfunction in a mouse model of high-fat diet-induced obesity

AJP Renal Physiology ◽

10.1152/ajprenal.00209.2013 ◽

2013 ◽

Vol 305 (6) ◽

pp. F871-F880 ◽

Cited By ~ 28

Author(s):

Wenpeng Cui ◽

Hasiyeti Maimaitiyiming ◽

Xinyu Qi ◽

Heather Norman ◽

Shuxia Wang

Keyword(s):

Mouse Model ◽

Renal Dysfunction ◽

Matrix Protein ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Kidney Diseases ◽

Extracellular Matrix Protein ◽

Obese Mouse ◽

Wild Type ◽

Thrombospondin 1

Obesity is prevalent worldwide and is a major risk factor for many diseases including renal complications. Thrombospondin 1 (TSP1), a multifunctional extracellular matrix protein, plays an important role in diabetic kidney diseases. However, whether TSP1 plays a role in obesity-related kidney disease is unknown. In the present studies, the role of TSP1 in obesity-induced renal dysfunction was determined by using a diet-induced obese mouse model. The results demonstrated that TSP1 was significantly upregulated in the kidney from obese mice. The increased TSP1 was localized in the glomerular mesangium as well as in the tubular system from obese wild-type mice. Obese wild-type mice developed renal hypertrophy and albuminuria, which was associated with increased kidney macrophage infiltration, augmented kidney inflammation, and activated transforming growth factor (TGF)-β signaling and renal fibrosis. In contrast, obese TSP1-deficient mice did not develop these kidney damages. Furthermore, in vitro studies demonstrated that leptin treatment stimulated the expression of TSP1, TGF-β1, fibronectin, and collagen type IV in mesangial cells isolated from wild-type mice. These leptin-stimulated effects were abolished in TSP1-deficient mesangial cells. Taken together, these data suggest that TSP1 is an important mediator for obesity- or hyperleptinemia-induced kidney dysfunction.

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Case Report: Occurrence of Severe Thoracic Aortic Aneurysms (Involving the Ascending, Arch, and Descending Segments) as a Result of Fibulin-4 Deficiency: A Rare Pathology With Successful Management

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.756765 ◽

2021 ◽

Vol 8 ◽

Author(s):

Paul Thomas ◽

Aparna Venugopalan ◽

Siddharth Narayanan ◽

Thomas Mathew ◽

Lakshmi Parvathi Deepti Cherukuwada ◽

...

Keyword(s):

Matrix Protein ◽

Genetic Disorder ◽

Female Child ◽

Aortic Aneurysms ◽

Cutis Laxa ◽

Extracellular Matrix Protein ◽

Successful Outcome ◽

High Morbidity ◽

Aortic Diseases ◽

Thoracic Aortic Aneurysms

Aortic diseases requiring surgery in childhood are distinctive and rare. Very few reports in the literature account for the occurrence of multiple thoracic aortic aneurysms in the same pediatric patient because of a genetic cause. We report a rare occurrence of severe thoracic aortic aneurysms (involving the ascending, arch and descending aortic segments) with severe aortic insufficiency in a 7-year-old female child secondary to the extremely rare and often lethal genetic disorder, cutis laxa. She was eventually identified as a carrier of a homozygous EFEMP2 (alias FBLN4) mutation. This gene encodes the extracellular matrix protein fibulin-4, and its mutation is associated with autosomal recessive cutis laxa type 1B that leads to severe aortopathy with aneurysm formation and vascular tortuosity. Parents of the child were not known to be consanguineous. Significant symptomatic improvement in the patient could be discerned after timely intervention with the valve-sparing aortic root replacement (David V procedure) and a concomitant aortic arch replacement. This is a unique report with a successful outcome that highlights the occurrence of a rare hereditary aortopathy associated with a high morbidity and mortality, and the importance of an early diagnosis and timely management. It also offers insight to physicians in having a very broad differential and multimodal approach in handling rare pediatric cardio-pathologies with a genetic predisposition.

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Abstract P139: Genetic Signature Of Accelerated Cardiac Fibrosis Due To Trpa1 Ablation

Hypertension ◽

10.1161/hyp.78.suppl_1.p139 ◽

2021 ◽

Vol 78 (Suppl_1) ◽

Author(s):

Shuangtao Ma ◽

Donna Wang

Keyword(s):

Matrix Metalloproteinases ◽

Matrix Protein ◽

Cardiac Fibrosis ◽

Collagen Fibers ◽

Extracellular Matrix Protein ◽

Myocardial Tissue ◽

Mrna Levels ◽

Wild Type ◽

Genetic Signature ◽

Age Related

While our previous study demonstrated that loss of transient receptor potential ankyrin 1 ( Trpa1 ) accelerates age-related cardiac fibrosis in mice, the underlying mechanism of potential anti-fibrotic property of TRPA1 remains largely unknown. TRPA1 is a sensor of oxidative stress and may play a protective role in age-related diseases. In this study, we performed quantitative polymerase chain reaction array analyses of the mRNA expression of 84 fibrosis-related genes in the myocardial tissue of 12-month-old Trpa1 -/- mice with significant cardiac fibrosis and age-matched wild-type mice without cardiac fibrosis. The mRNA levels of Col1a2 and Col3a1 in the myocardial tissue were similar between Trpa1 -/- and wild-type mice, suggesting comparable cardiac collagen synthesis in the two strains. Matrix metalloproteinases are major enzymes responsible for degradation of collagen fibers. The results show that the mRNA levels of matrix metalloproteinases, including Mmp1a , Mmp2 , Mmp3 , Mmp8 , Mmp9 , Mmp13 , and Mmp14 , in the heart were similar between Trpa1 -/- and wild-type mice. Nevertheless, we identified 7 significantly changed genes in the heart between the two strains. The expression levels of Acta2 , Inhbe , Ifng , and Ccl11 were significantly increased with fold changes of 3.1, 1.9, 1.9, and 1.5 (all P < 0.05), respectively, while Timp3 , Stat6 , and Ilk were significantly decreased with fold changes of 0.3, 0.5, and 0.7 (all P < 0.05), respectively, in the heart of Trpa1 -/- mice compared with wild-type mice. Acta2 , the most upregulated gene in Trpa1 -/- hearts, is a marker of myofibroblasts. Its upregulation indicates increased differentiation from fibroblasts into myofibroblasts in Trpa1 -/- hearts compared with wild-type hearts. Timp3 , the most downregulated gene in Trpa1 -/- hearts, codes an extracellular matrix protein TIMP3, which not only inhibits matrix metalloproteinases but also regulate post-translational modification of collagen fibers. Taken together, these findings suggest that upregulation of Acta2 and downregulation of Timp3 may serve as genetic signature or play a role in accelerated age-related cardiac fibrosis due to TRPA1 ablation.

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Mutant Dentin Sialophosphoprotein Causes Dentinogenesis Imperfecta

Journal of Dental Research ◽

10.1177/0022034519854029 ◽

2019 ◽

Vol 98 (8) ◽

pp. 912-919 ◽

Cited By ~ 4

Author(s):

T. Liang ◽

H. Zhang ◽

Q. Xu ◽

S. Wang ◽

C. Qin ◽

...

Keyword(s):

Dental Pulp ◽

Matrix Protein ◽

Mutant Mice ◽

Extracellular Matrix Protein ◽

Dentinogenesis Imperfecta ◽

Wild Type ◽

Pulp Chamber ◽

Dentin Sialophosphoprotein ◽

In The Wild

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein highly expressed by odontoblasts in teeth. DSPP mutations in humans may cause dentinogenesis imperfecta (DGI), an autosomal dominant dentin disorder. We recently generated a mouse model (named “ DsppP19L/+ mice”) that expressed a mutant DSPP in which the proline residue at position 19 was replaced by a leucine residue. We found that the DsppP19L/+ and DsppP19L/P19L mice at a younger age displayed a tooth phenotype resembling human DGI type III characterized by enlarged dental pulp chambers, while the teeth of older DsppP19L/+ and DsppP19L/P19L mice had smaller dental pulp chambers mimicking DGI type II. The teeth of DsppP19L/+ and DsppP19L/P19L mice had a narrower pulp chamber roof predentin layer, thinner pulp chamber roof dentin, and thicker pulp chamber floor dentin. In addition, these mice also had increased enamel attrition, accompanied by excessive deposition of peritubular dentin. Immunohistochemistry, in situ hybridization, and real-time polymerase chain reaction analyses showed that the odontoblasts in both DsppP19L/+ and DsppP19L/P19L mice had reduced DSPP expression, compared to the wild-type mice. We also observed that the levels of DSPP expression were much higher in the roof-forming odontoblasts than in the floor-forming odontoblasts in the wild-type mice and mutant mice. Moreover, immunohistochemistry showed that while the immunostaining signals of dentin sialoprotein (N-terminal fragment of DSPP) were decreased in the dentin matrix, they were remarkably increased in the odontoblasts of the DsppP19L/+ and DsppP19L/P19L mice. Consistently, our in vitro studies showed that the secretion of the mutant DSPP was impaired and accumulated within endoplasmic reticulum. These findings suggest that the dental phenotypes of the mutant mice were associated with the intracellular retention of the mutant DSPP in the odontoblasts of the DSPP-mutant mice.

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Differences in genetic signaling, and not mechanical properties of the wall, are linked to ascending aortic aneurysms in fibulin-4 knockout mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00178.2015 ◽

2015 ◽

Vol 309 (1) ◽

pp. H103-H113 ◽

Cited By ~ 8

Author(s):

Jungsil Kim ◽

Jesse D. Procknow ◽

Hiromi Yanagisawa ◽

Jessica E. Wagenseil

Keyword(s):

Gene Expression ◽

Matrix Protein ◽

Mechanical Response ◽

Aortic Aneurysms ◽

Extracellular Matrix Protein ◽

Elastic Fibers ◽

Newborn Mice ◽

Thoracic Aortic Aneurysms ◽

Neutrophil Collagenase

Fibulin-4 is an extracellular matrix protein that is essential for proper assembly of arterial elastic fibers. Mutations in fibulin-4 cause cutis laxa with thoracic aortic aneurysms (TAAs). Sixty percent of TAAs occur in the ascending aorta (AA). Newborn mice lacking fibulin-4 ( Fbln4−/−) have aneurysms in the AA, but narrowing in the descending aorta (DA), and are a unique model to investigate locational differences in aneurysm susceptibility. We measured mechanical behavior and gene expression of AA and DA segments in newborn Fbln4−/− and Fbln4+/+ mice. Fbln4−/− AA has increased diameters compared with Fbln4+/+ AA and Fbln4−/− DA at most applied pressures, confirming genotypic and locational specificity of the aneurysm phenotype. When diameter compliance and tangent modulus were calculated from the mechanical data, we found few significant differences between genotypes, suggesting that the mechanical response to incremental diameter changes is similar, despite the fragmented elastic fibers in Fbln4−/− aortas. Fbln4−/− aortas showed a trend toward increased circumferential stretch, which may be transmitted to smooth muscle cells (SMCs) in the wall. Gene expression data suggest activation of pathways for SMC proliferation and inflammation in Fbln4−/− aortas compared with Fbln4+/+. Additional genes in both pathways, as well as matrix metalloprotease-8 ( Mmp8), are upregulated specifically in Fbln4−/− AA compared with Fbln4+/+ AA and Fbln4−/− DA. Mmp8 is a neutrophil collagenase that targets type 1 collagen, and upregulation may be necessary to allow diameter expansion in Fbln4−/− AA. Our results provide molecular and mechanical targets for further investigation in aneurysm pathogenesis.

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Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans

Infection and Immunity ◽

10.1128/iai.01370-08 ◽

2008 ◽

Vol 77 (3) ◽

pp. 952-958 ◽

Cited By ~ 77

Author(s):

Gerald L. Murray ◽

Amporn Srikram ◽

David E. Hoke ◽

Elsio A. Wunder ◽

Rebekah Henry ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Chronic Infection ◽

Matrix Protein ◽

Severe Disease ◽

Extracellular Matrix Protein ◽

Leptospira Interrogans ◽

Renal Tubules ◽

Wild Type

ABSTRACT Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B12. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.

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Experimental Inhibition of Periostin Attenuates Kidney Fibrosis

American Journal of Nephrology ◽

10.1159/000485325 ◽

2017 ◽

Vol 46 (6) ◽

pp. 501-517 ◽

Cited By ~ 7

Author(s):

Jin Ho Hwang ◽

Seung Hee Yang ◽

Yong Chul Kim ◽

Jin Hyuk Kim ◽

Jung Nam An ◽

...

Keyword(s):

Renal Fibrosis ◽

Matrix Protein ◽

Collecting Duct ◽

Extracellular Matrix Protein ◽

In Vitro Experiments ◽

Wild Type ◽

In The Wild ◽

Renal Fibrogenesis

Background: Periostin is responsible for tissue regeneration, fibrosis, and wound healing via its interaction with integrin. Recently, the role of periostin has been shown to contribute to fibrosis in chronic kidney disease. We investigated the role of periostin and the effect of periostin blockade in renal fibrogenesis. Methods: We investigated the function of periostin in vivo in wild-type and periostin-null mice (Postn-KO) in a unilateral ureteral obstruction (UUO) model. For the in vitro experiments, primary cultured inner medullary collecting duct cells from the wild-type and Postn-KO mice were used. Results: Periostin expression was strongly induced by UUO in the wild-type mice. UUO induced renal fibrosis and morphological changes in the obstructed kidney of wild-type mice, whereas global knockout of periostin reduced fibrosis induced by UUO and improved kidney structure. Fibrosis- and inflammation-related mRNA were significantly induced in the wild-type mice and were decreased in the Postn-KO mice. Additionally, α-smooth muscle actin expression was increased following the administration of recombinant periostin in vitro. The effect of periostin blockade was examined using 2 methods. The integrin blockade peptide decreased fibrosis-related gene expression in in vitro experiments. Anti-periostin polyclonal antibody attenuated renal fibrosis induced by UUO through changes in transforming growth factor-β signaling and the inflammatory and apoptotic pathways. Conclusion: Periostin is a marker of renal fibrosis and may augment the progression of fibrogenesis as an extracellular matrix protein. Periostin blockade effectively attenuated renal fibrogenesis. Thus, periostin inhibition may be a therapeutic strategy for the amelioration of renal disease progression.

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