A thrombospondin-dependent pathway for a protective ER stress response.

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

Disruption of valosin-containing protein activity causes cardiomyopathy and reveals pleiotropic functions in cardiac homeostasis.

Valosin-containing protein (VCP), also known as p97, is an ATPase with diverse cellular functions, although the most highly characterized is targeting of misfolded or aggregated proteins to degradation pathways, including the endoplasmic reticulum-associated degradation (ERAD) pathway. However, how VCP functions in the heart has not been carefully examined despite the fact that human mutations in VCP cause Paget disease of bone and frontotemporal dementia, an autosomal dominant multisystem proteinopathy that includes disease in the heart, skeletal muscle, brain, and bone. Here we generated heart-specific transgenic mice overexpressing WT VCP or a VCPK524A mutant with deficient ATPase activity. Transgenic mice overexpressing WT VCP exhibit normal cardiac structure and function, whereas mutant VCP-overexpressing mice develop cardiomyopathy. Mechanistically, mutant VCP-overexpressing hearts up-regulate ERAD complex components and have elevated levels of ubiquitinated proteins prior to manifestation of cardiomyopathy, suggesting dysregulation of ERAD and inefficient clearance of proteins targeted for proteasomal degradation. The hearts of mutant VCP transgenic mice also exhibit profound defects in cardiomyocyte nuclear morphology with increased nuclear envelope proteins and nuclear lamins. Proteomics revealed overwhelming interactions of endogenous VCP with ribosomal, ribosome-associated, and RNA-binding proteins in the heart, and impairment of cardiac VCP activity resulted in aggregation of large ribosomal subunit proteins. These data identify multifactorial functions and diverse mechanisms whereby VCP regulates cardiomyocyte protein and RNA quality control that are critical for cardiac homeostasis, suggesting how human VCP mutations negatively affect the heart.

Acute Catecholamine Exposure Causes Reversible Myocyte Injury Without Cardiac Regeneration.

RATIONALE: Catecholamines increase cardiac contractility, but exposure to high concentrations or prolonged exposures can cause cardiac injury. A recent study demonstrated that a single subcutaneous injection of isoproterenol (ISO; 200 mg/kg) in mice causes acute myocyte death (8%-10%) with complete cardiac repair within a month. Cardiac regeneration was via endogenous cKit(+) cardiac stem cell-mediated new myocyte formation. OBJECTIVE: Our goal was to validate this simple injury/regeneration system and use it to study the biology of newly forming adult cardiac myocytes. METHODS AND RESULTS: C57BL/6 mice (n=173) were treated with single injections of vehicle, 200 or 300 mg/kg ISO, or 2 daily doses of 200 mg/kg ISO for 6 days. Echocardiography revealed transiently increased systolic function and unaltered diastolic function 1 day after single ISO injection. Single ISO injections also caused membrane injury in ≈10% of myocytes, but few of these myocytes appeared to be necrotic. Circulating troponin I levels after ISO were elevated, further documenting myocyte damage. However, myocyte apoptosis was not increased after ISO injury. Heart weight to body weight ratio and fibrosis were also not altered 28 days after ISO injection. Single- or multiple-dose ISO injury was not associated with an increase in the percentage of 5-ethynyl-2'-deoxyuridine-labeled myocytes. Furthermore, ISO injections did not increase new myocytes in cKit(+/Cre)×R-GFP transgenic mice. CONCLUSIONS: A single dose of ISO causes injury in ≈10% of the cardiomyocytes. However, most of these myocytes seem to recover and do not elicit cKit(+) cardiac stem cell-derived myocyte regeneration.

Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and ß-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD.

Osteoglycin prevents cardiac dilatation and dysfunction after myocardial infarction through infarct collagen strengthening.

RATIONALE: To maintain cardiac mechanical and structural integrity after an ischemic insult, profound alterations occur within the extracellular matrix. Osteoglycin is a small leucine-rich proteoglycan previously described as a marker of cardiac hypertrophy. OBJECTIVE: To establish whether osteoglycin may play a role in cardiac integrity and function after myocardial infarction (MI). METHODS AND RESULTS: Osteoglycin expression is associated with collagen deposition and scar formation in mouse and human MI. Absence of osteoglycin in mice resulted in significantly increased rupture-related mortality with tissue disruption, intramyocardial bleeding, and increased cardiac dysfunction, despite equal infarct sizes. Surviving osteoglycin null mice had greater infarct expansion in comparison with wild-type mice because of impaired collagen fibrillogenesis and maturation in the infarcts as revealed by electron microscopy and collagen polarization. Absence of osteoglycin did not affect cardiomyocyte hypertrophy in the remodeling remote myocardium. In cultured fibroblasts, osteoglycin knockdown or supplementation did not alter transforming growth factor-ß signaling. Adenoviral overexpression of osteoglycin in wild-type mice significantly improved collagen quality, thereby blunting cardiac dilatation and dysfunction after MI. In osteoglycin null mice, adenoviral overexpression of osteoglycin was unable to prevent rupture-related mortality because of insufficiently restoring osteoglycin protein levels in the heart. Finally, circulating osteoglycin levels in patients with heart failure were significantly increased in the patients with a previous history of MI compared with those with nonischemic heart failure and correlated with survival, left ventricular volumes, and other markers of fibrosis. CONCLUSIONS: Increased osteoglycin expression in the infarct scar promotes proper collagen maturation and protects against cardiac disruption and adverse remodeling after MI. In human heart failure, osteoglycin is a promising biomarker for ischemic heart failure.

Deletion of Periostin Protects Against Atherosclerosis in Mice by Altering Inflammation and Extracellular Matrix Remodeling.

OBJECTIVE: Periostin is a secreted protein that can alter extracellular matrix remodeling in response to tissue injury. However, the functional role of periostin in the development of atherosclerotic plaques has yet to be described despite its observed induction in diseased vessels and presence in the serum. APPROACH AND RESULTS: Hyperlipidemic, apolipoprotein E-null mice (ApoE(-/) (-)) were crossed with periostin (Postn(-/-)) gene-deleted mice and placed on a high-fat diet for 6 or 14 weeks to induce atherosclerosis. En face analysis of aortas showed significantly decreased lesion areas of ApoE(-/-) Postn(-/-) mice compared with ApoE(-/-) mice, as well as a reduced inflammatory response with less macrophage content. Moreover, diseased aortas from ApoE(-/-) Postn(-/-) mice displayed a disorganized extracellular matrix with less collagen cross linking and smaller fibrotic caps, as well as increased matrix metalloproteinase-2, metalloproteinase-13, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase-1 mRNA expression. Furthermore, the loss of periostin was associated with a switch in vascular smooth muscle cells toward a more proliferative and synthetic phenotype. Mechanistically, the loss of periostin reduced macrophage recruitment by transforming growth factor-ß in cellular migration assays. CONCLUSIONS: These are the first genetic data detailing the function of periostin as a regulator of atherosclerotic lesion formation and progression. The data suggest that periostin could be a therapeutic target for atherosclerotic plaque formation through modulation of the immune response and extracellular matrix remodeling.

Cardiomyocyte-specific transforming growth factor ß suppression blocks neutrophil infiltration, augments multiple cytoprotective cascades, and reduces early mortality after myocardial infarction.

RATIONALE: Wound healing after myocardial infarction involves a highly regulated inflammatory response that is initiated by the appearance of neutrophils to clear out dead cells and matrix debris. Neutrophil infiltration is controlled by multiple secreted factors, including the master regulator transforming growth factor ß (TGFß). Broad inhibition of TGFß early postinfarction has worsened post-myocardial infarction remodeling; however, this signaling displays potent cell specificity, and targeted suppression particularly in the myocyte could be beneficial. OBJECTIVE: Our aims were to test the hypothesis that targeted suppression of myocyte TGFß signaling ameliorates postinfarct remodeling and inflammatory modulation and to identify mechanisms by which this may be achieved. METHODS AND RESULTS: Mice with TGFß receptor-coupled signaling genetically suppressed only in cardiac myocytes (conditional TGFß receptor 1 or 2 knockout) displayed marked declines in neutrophil recruitment and accompanying metalloproteinase 9 activation after infarction and were protected against early-onset mortality due to wall rupture. This is a cell-specific effect, because broader inhibition of TGFß signaling led to 100% early mortality due to rupture. Rather than by altering fibrosis or reducing the generation of proinflammatory cytokines/chemokines, myocyte-selective TGFß inhibition augmented the synthesis of a constellation of highly protective cardiokines. These included thrombospondin 4 with associated endoplasmic reticulum stress responses, interleukin-33, follistatin-like 1, and growth and differentiation factor 15, which is an inhibitor of neutrophil integrin activation and tissue migration. CONCLUSIONS: These data reveal a novel role of myocyte TGFß signaling as a potent regulator of protective cardiokine and neutrophil-mediated infarct remodeling.

Matricellular proteins and matrix metalloproteinases mark the inflammatory and fibrotic response in human cardiac allograft rejection.

AIMS: The cardiac extracellular matrix is highly involved in regulating inflammation, remodelling, and function of the heart. Whether matrix alterations relate to the degree of inflammation, fibrosis, and overall rejection in the human transplanted heart remained, until now, unknown. METHODS AND RESULTS: Expression of matricellular proteins, proteoglycans, and metalloproteinases (MMPs) and their inhibitors (TIMPs) were investigated in serial endomyocardial biopsies (n = 102), in a cohort of 39 patients within the first year after cardiac transplantation. Out of 15 matrix-related proteins, intragraft transcript and protein levels of syndecan-1 and MMP-9 showed a strong association with the degree of cardiac allograft rejection (CAR), the expression of pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6 and transforming growth factor (TGF)-ß, and with infiltrating CD3⁺ T-cells and CD68⁺ monocytes. In addition, SPARC, CTGF, TSP-2, MMP-14, TIMP-1, Testican-1, TSP-1, Syndecan-1, MMP-2, -9, and -14, as well as IL-6 and TGF-ß transcript levels and inflammatory infiltrates all strongly relate to collagen expression in the transplanted heart. More importantly, receiver operating characteristic curve analysis demonstrated that syndecan-1 and MMP-9 transcript levels had the highest area under the curve (0.969 and 0.981, respectively), thereby identifying both as a potential decision-making tool to discriminate rejecting from non-rejecting hearts. CONCLUSION: Out of 15 matrix-related proteins, we identified synd-1 and MMP-9 intragraft transcript levels of as strong predictors of human CAR. In addition, a multitude of non-structural matrix-related proteins closely associate with collagen expression in the transplanted heart. Therefore, we are convinced that these findings deserve further investigation and are likely to be of clinical value to prevent human CAR.

Thbs1 regulates skeletal muscle mass in a TGFß-Smad2/3-ATF4-dependent manner.

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor ß (TGFß)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFß-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFß-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFß-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.

Gucy1α1 specifically marks kidney, heart, lung and liver fibroblasts.

Fibrosis is a common outcome of numerous pathologies, including chronic kidney disease (CKD), a progressive renal function deterioration. Current approaches to target activated fibroblasts, key effector contributors to fibrotic tissue remodeling, lack specificity. Here, we report Gucy1α1 as a specific kidney fibroblast marker. Gucy1α1 levels significantly increased over the course of two clinically relevant murine CKD models and directly correlated with established fibrosis markers. Immunofluorescent (IF) imaging showed that Gucy1α1 comprehensively labelled cortical and medullary quiescent and activated fibroblasts in the control kidney and throughout injury progression, respectively. Unlike traditionally used markers platelet derived growth factor receptor beta (Pdgfrß) and vimentin (Vim), Gucy1α1 did not overlap with off-target populations such as podocytes. Notably, Gucy1α1 labelled kidney fibroblasts in both male and female mice. Furthermore, we observed elevated GUCY1α1 expression in the human fibrotic kidney and lung. Studies in the murine models of cardiac and liver fibrosis revealed Gucy1α1 elevation in activated Pdgfrß-, Vim- and alpha smooth muscle actin (αSma)-expressing fibroblasts paralleling injury progression and resolution. Overall, we demonstrate Gucy1α1 as an exclusive fibroblast marker in both sexes. Due to its multiorgan translational potential, GUCY1α1 might provide a novel promising strategy to specifically target and mechanistically examine fibroblasts.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

MCUb is an inducible regulator of calcium-dependent mitochondrial metabolism and substrate utilization in muscle.

Mitochondria use the electron transport chain to generate high-energy phosphate from oxidative phosphorylation, a process also regulated by the mitochondrial Ca2+ uniporter (MCU) and Ca2+ levels. Here, we show that MCUb, an inhibitor of MCU-mediated Ca2+ influx, is induced by caloric restriction, where it increases mitochondrial fatty acid utilization. To mimic the fasted state with reduced mitochondrial Ca2+ influx, we generated genetically altered mice with skeletal muscle-specific MCUb expression that showed greater fatty acid usage, less fat accumulation, and lower body weight. In contrast, mice lacking Mcub in skeletal muscle showed increased pyruvate dehydrogenase activity, increased muscle malonyl coenzyme A (CoA), reduced fatty acid utilization, glucose intolerance, and increased adiposity. Mechanistically, pyruvate dehydrogenase kinase 4 (PDK4) overexpression in muscle of Mcub-deleted mice abolished altered substrate preference. Thus, MCUb is an inducible control point in regulating skeletal muscle mitochondrial Ca2+ levels and substrate utilization that impacts total metabolic balance.

Rpl3l gene deletion in mice reduces heart weight over time.

Introduction: The ribosomal protein L3-like (RPL3L) is a heart and skeletal muscle-specific ribosomal protein and paralogue of the more ubiquitously expressed RPL3 protein. Mutations in the human RPL3L gene are linked to childhood cardiomyopathy and age-related atrial fibrillation, yet the function of RPL3L in the mammalian heart remains unknown. Methods and Results: Here, we observed that mouse cardiac ventricles express RPL3 at birth, where it is gradually replaced by RPL3L in adulthood but re-expressed with induction of hypertrophy in adults. Rpl3l gene-deleted mice were generated to examine the role of this gene in the heart, although Rpl3l -/- mice showed no overt changes in cardiac structure or function at baseline or after pressure overload hypertrophy, likely because RPL3 expression was upregulated and maintained in adulthood. mRNA expression analysis and ribosome profiling failed to show differences between the hearts of Rpl3l null and wild type mice in adulthood. Moreover, ribosomes lacking RPL3L showed no differences in localization within cardiomyocytes compared to wild type controls, nor was there an alteration in cardiac tissue ultrastructure or mitochondrial function in adult Rpl3l -/- mice. Similarly, overexpression of either RPL3 or RPL3L with adeno-associated virus -9 in the hearts of mice did not cause discernable pathology. However, by 18 months of age Rpl3l -/- null mice had significantly smaller hearts compared to wild type littermates. Conclusion: Thus, deletion of Rpl3l forces maintenance of RPL3 expression within the heart that appears to fully compensate for the loss of RPL3L, although older Rpl3l -/- mice showed a mild but significant reduction in heart weight.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8, while the surviving proximal tubules (PTs) showed restored transcriptional signature. Furthermore, we found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Malignant cells fuel tumor growth by educating infiltrating leukocytes to produce the mitogen Gas6.

The transforming and tumor growth-promoting properties of Axl, a member of the Tyro3, Axl, and Mer (TAM) family of receptor tyrosine kinases (TAMRs), are well recognized. In contrast, little is known about the role of the TAMR ligand growth arrest-specific gene 6 (Gas6) in tumor biology. By using Gas6-deficient (Gas6(-/-)) mice, we show that bone marrow-derived Gas6 promotes growth and metastasis in different experimental cancer models, including one resistant to vascular endothelial growth factor inhibitors. Mechanistic studies reveal that circulating leukocytes produce minimal Gas6. However, once infiltrated in the tumor, leukocytes up-regulate Gas6, which is mitogenic for tumor cells. Consistent herewith, impaired tumor growth in Gas6(-/-) mice is rescued by transplantation of wild-type bone marrow and, conversely, mimicked by transplantation of Gas6(-/-) bone marrow into wild-type hosts. These findings highlight a novel role for Gas6 in a positive amplification loop, whereby tumors promote their growth by educating infiltrating leukocytes to up-regulate the production of the mitogen Gas6. Hence, inhibition of Gas6 might offer novel opportunities for the treatment of cancer.

The matrix in focus: new directions in extracellular matrix research from the 2021 ASMB hybrid meeting.

The extracellular matrix (ECM) is a complex assembly of macromolecules that provides both architectural support and molecular signals to cells and modulate their behaviors. Originally considered a passive mechanical structure, decades of research have since demonstrated how the ECM dynamically regulates a diverse set of cellular processes in development, homeostasis, and disease progression. In September 2021, the American Society for Matrix Biology (ASMB) organized a hybrid scientific meeting, integrating in-person and virtual formats, to discuss the latest developments in ECM research. Here, we highlight exciting scientific advances that emerged from the meeting including (1) the use of model systems for fundamental and translation ECM research, (2) ECM-targeting approaches as therapeutic modalities, (3) cell-ECM interactions, and (4) the ECM as a critical component of tissue engineering strategies. In addition, we discuss how the ASMB incorporated mentoring, career development, and diversity, equity, and inclusion initiatives in both virtual and in-person events. Finally, we reflect on the hybrid scientific conference format and how it will help the ASMB accomplish its mission moving forward.

Matricellular signaling molecule CCN1 attenuates experimental autoimmune myocarditis by acting as a novel immune cell migration modulator.

BACKGROUND: CCN1 is an evolutionary ancient matricellular protein that modulates biological processes associated with tissue repair. Induction at sites of injury was observed in conditions ranging from skin wounds to cardiac diseases, including ischemic and inflammatory cardiomyopathy. Here, we provide evidence of a novel function of CCN1 as a modulator of immune cell migration. METHODS AND RESULTS: to understand the role of CCN1 in cardiomyopathies and to evaluate its therapeutic potential, we overexpressed CCN1 using an adenoviral hepatotropic vector in murine experimental autoimmune myocarditis, a model of human inflammatory cardiomyopathy. CCN1 gene transfer significantly reduced cardiac disease score and immune cell infiltration. In vivo tracking of hemagglutinin epitope-tagged CCN1 revealed binding to spleen macrophages but not to cardiomyocytes. Unexpectedly, CCN1 therapy left cardiac chemokine and cytokine expression unchanged but instead strongly inhibited the migration of spleen macrophages and lymphocytes, as evidenced by ex vivo transwell assays. In accordance with the ex vivo data, in vitro preincubation with CCN1 diminished transwell migration of human monocytes and abrogated their chemotactic response to monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and stromal cell-derived factor-1α. Further mechanistic studies showed that CCN1-driven modulation of immune cell migration is mimicked in part by cyclic RGD peptides currently in clinical evaluation for cancer therapy. CONCLUSIONS: our proof-of-concept study suggests investigation of CCN1 as a novel, endogenous "parent compound" for chemotaxis modulation and of cyclic RGD peptides as a class of partially CCN1-mimetic drugs with immediate potential for clinical evaluation in cardiac diseases associated with chronic pathogenic inflammation.

Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy.

The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1-/- mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart.

TIMPs and cardiac remodeling: 'Embracing the MMP-independent-side of the family'.

Unraveling the biological role of tissue inhibitors of metalloproteinases (TIMPs) during cardiac remodeling and the progression of heart failure has proven to be an enormous challenge. Remodeling of the cardiac extracellular matrix (ECM), regulated by matrix metalloproteinases (MMPs) and their endogenous inhibitors, TIMPs, is a well-established paradigm in cardiac health and disease. Originally, TIMPs were thought to function exclusively as endogenous inhibitors of MMP activity, thereby fine-tuning MMP-mediated ECM degradation and numerous related processes. However, during the last two decades, the concept of MMP-independent TIMP-mediated receptor signaling and regulation of cell fate has emerged. Although our current knowledge is still limited, in this review, we highlight some of the novel data, illustrating the MMP-independent biological properties of the four TIMP family members. Moreover, we discuss how these cell-specific insights may contribute to the process of cardiac remodeling, disease and failure. Finally, we identify where additional research is needed that will codetermine the possible future of TIMPs as therapeutic targets.

Absence of thrombospondin-2 causes age-related dilated cardiomyopathy.

BACKGROUND: The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart. METHODS AND RESULTS: TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas <10% of the wild-type mice died. In the absence of TSP-2, older mice displayed a severe dilated cardiomyopathy with impaired systolic function, increased cardiac dilatation, and fibrosis. Ultrastructural analysis revealed progressive myocyte stress and death, accompanied by an inflammatory response and replacement fibrosis, in aging TSP-2 KO animals, whereas capillary or coronary morphology or density was not affected. Importantly, adeno-associated virus-9 gene-mediated transfer of TSP-2 in 7-week-old TSP-2 KO mice normalized their survival and prevented dilated cardiomyopathy. In TSP-2 KO animals, age-related cardiomyopathy was accompanied by increased matrix metalloproteinase-2 and decreased tissue transglutaminase-2 activity, together with impaired collagen cross-linking. At the cardiomyocyte level, TSP-2 deficiency in vivo and its knockdown in vitro decreased the activation of the Akt survival pathway in cardiomyocytes. CONCLUSIONS: TSP-2 expression in the heart protects against age-dependent dilated cardiomyopathy.