Systems pharmacology-based integration of human and mouse data for drug repurposing to treat thoracic aneurysms.

Marfan syndrome (MFS) is associated with mutations in fibrillin-1 that predispose afflicted individuals to progressive thoracic aortic aneurysm (TAA) leading to dissection and rupture of the vessel wall. Here we combined computational and experimental approaches to identify and test FDA-approved drugs that may slow or even halt aneurysm progression. Computational analyses of transcriptomic data derived from the aortas of MFS patients and MFS mice (Fbn1mgR/mgR mice) predicted that subcellular pathways associated with reduced muscle contractility are key TAA determinants that could be targeted with the GABAB receptor agonist baclofen. Systemic administration of baclofen to Fbn1mgR/mgR mice validated our computational prediction by mitigating arterial disease progression at the cellular and physiological levels. Interestingly, baclofen improved muscle contraction-related subcellular pathways by upregulating a different set of genes than those downregulated in the aorta of vehicle-treated Fbn1mgR/mgR mice. Distinct transcriptomic profiles were also associated with drug-treated MFS and wild-type mice. Thus, systems pharmacology approaches that compare patient- and mouse-derived transcriptomic data for subcellular pathway-based drug repurposing represent an effective strategy to identify potential new treatments of human diseases.

Massive aggrecan and versican accumulation in thoracic aortic aneurysm and dissection.

Proteoglycan accumulation is a hallmark of medial degeneration in thoracic aortic aneurysm and dissection (TAAD). Here, we defined the aortic proteoglycanome using mass spectrometry, and based on the findings, investigated the large aggregating proteoglycans aggrecan and versican in human ascending TAAD and a mouse model of severe Marfan syndrome. The aortic proteoglycanome comprises 20 proteoglycans including aggrecan and versican. Antibodies against these proteoglycans intensely stained medial degeneration lesions in TAAD, contrasting with modest intralamellar staining in controls. Aggrecan, but not versican, was increased in longitudinal analysis of Fbn1mgR/mgR aortas. TAAD and Fbn1mgR/mgR aortas had increased aggrecan and versican mRNAs, and reduced expression of a key proteoglycanase gene, ADAMTS5, was seen in TAAD. Fbn1mgR/mgR mice with ascending aortic dissection and/or rupture had dramatically increased aggrecan staining compared with mice without these complications. Thus, aggrecan and versican accumulation in ascending TAAD occurs via increased synthesis and/or reduced proteolytic turnover, and correlates with aortic dissection/rupture in Fbn1mgR/mgR mice. Tissue swelling imposed by aggrecan and versican is proposed to be profoundly deleterious to aortic wall mechanics and smooth muscle cell homeostasis, predisposing to type-A dissections. These proteoglycans provide potential biomarkers for refined risk stratification and timing of elective aortic aneurysm repair.

Cell Type-Specific Contributions of the Angiotensin II Type 1a Receptor to Aorta Homeostasis and Aneurysmal Disease-Brief Report.

OBJECTIVE: Two were the aims of this study: first, to translate whole-genome expression profiles into computational predictions of functional associations between signaling pathways that regulate aorta homeostasis and the activity of angiotensin II type 1a receptor (At1ar) in either vascular endothelial or smooth muscle cells; and second, to characterize the impact of endothelial cell- or smooth muscle cell-specific At1ar disruption on the development of thoracic aortic aneurysm in fibrillin-1 hypomorphic (Fbn1mgR/mgR ) mice, a validated animal model of early onset progressively severe Marfan syndrome. APPROACH AND RESULTS: Cdh5-Cre and Sm22-Cre transgenic mice were used to inactivate the At1ar-coding gene (Agt1ar) in either intimal or medial cells of both wild type and Marfan syndrome mice, respectively. Computational analyses of differentially expressed genes predicted dysregulated signaling pathways of cell survival and matrix remodeling in Agt1arCdh5-/- aortas and of cell adhesion and contractility in Agt1arSm22-/- aortas. Characterization of Fbn1mgR/mgR;Agt1arCdh5-/- mice revealed increased median survival associated with mitigated aneurysm growth and media degeneration, as well as reduced levels of phosphorylated (p-) Erk1/2 but not p-Smad2. By contrast, levels of both p-Erk1/2 and p-Smad2 proteins were normalized in Fbn1mgR/mgR;Agt1arSm22-/- aortas in spite of them showing no appreciable changes in thoracic aortic aneurysm pathology. CONCLUSIONS: Physiological At1ar signaling in the intimal and medial layers is associated with distinct regulatory processes of aorta homeostasis and function; improper At1ar activity in the vascular endothelium is a significant determinant of thoracic aortic aneurysm development in Marfan syndrome mice.

Marfan syndrome; A connective tissue disease at the crossroads of mechanotransduction, TGFß signaling and cell stemness.

Mutations in fibrillin-1 cause Marfan syndrome (MFS), the most common heritable disorder of connective tissue. Fibrillin-1 assemblies (microfibrils and elastic fibers) represent a unique dual-function component of the architectural matrix. The first role is structural for they endow tissues with tensile strength and elasticity, transmit forces across them and demarcate functionally discrete areas within them. The second role is instructive in that these macroaggregates modulate a large variety of sub-cellular processes by interacting with mechanosensors, and integrin and syndecan receptors, and by modulating the bioavailability of local TGFß signals. The multifunctional, tissue-specific nature of fibrillin-1 assemblies is reflected in the variety of clinical manifestations and disease mechanisms associated with the MFS phenotype. Characterization of mice with ubiquitous or cell type-restricted fibrillin-1 deficiency has unraveled some pathophysiological mechanisms associated with the MFS phenotype, such as altered mechanotransduction in the heart, dysregulated TGFß signaling in the ascending aorta and perturbed stem cell fate in the bone marrow. In each case, potential druggable targets have also been identified. However, the finding that distinct disease mechanisms underlie different organ abnormalities strongly argues for developing multi-drug strategies to mitigate or even prevent both life-threatening and morbid manifestations in pediatric and adult MFS patients.

Losartan Attenuates Degradation of Aorta and Lung Tissue Micromechanics in a Mouse Model of Severe Marfan Syndrome.

Marfan syndrome (MFS) is an autosomal dominant disease of the connective tissue due to mutations in the fibrillin-1 gene (FBN1). This study aimed at characterizing microelastic properties of the ascending aortic wall and lung parenchyma tissues from wild type (WT) and age-matched Fbn1 hypomorphic mice (Fbn1(mgR/mgR) mice) to identify tissue-specific biomechanical effects of aging and disease in MFS. Atomic force microscopy was used to indent lung parenchyma and aortic wall tissues, using Hybrid Eshelby Decomposition analysis to extract layer-specific properties of the intima and media. The intima stiffened with age and was not different between WT and Fbn1(mgR/mgR) tissues, whereas the media layer of MFS aortas showed progressive structural and mechanical degradation with a modulus that was 50% softer than WT by 3.5 months of age. Similarly, MFS mice displayed progressive structural and mechanical deterioration of lung tissue, which was over 85% softer than WT by 3.5 months of age. Chronic treatment with the angiotensin type I receptor antagonist, losartan, attenuated the aorta and lung tissue degradation, resulting in structural and mechanical properties not significantly different from age-matched WT controls. By revealing micromechanical softening of elastin-rich aorta and lung tissues with disease progression in fibrillin-1 deficient mice, our findings support the use of losartan as a prophylactic treatment that may abrogate the life-threatening symptoms of MFS.

Dimorphic effects of transforming growth factor-ß signaling during aortic aneurysm progression in mice suggest a combinatorial therapy for Marfan syndrome.

OBJECTIVE: Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of noncanonical (Erk-mediated) TGFß signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFß neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFß neutralization and AT1r antagonism in mice with progressively severe MFS (Fbn1(mgR/mgR) mice). APPROACH AND RESULTS: Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1(mgR/mgR) mice were compared after a ≈3-month-long treatment with placebo and either the AT1r antagonist losartan or the TGFß-neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFß neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (postnatal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence-related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFß hyperactivity that was in part AT1r dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1(mgR/mgR) mice from ruptured TAA. CONCLUSIONS: By demonstrating that promiscuous AT1r and TGFß drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways although sparing the early protective role of TGFß.

Cardiac lineage protein-1 (CLP-1) regulates cardiac remodeling via transcriptional modulation of diverse hypertrophic and fibrotic responses and angiotensin II-transforming growth factor ß (TGF-ß1) signaling axis.

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-ß1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-ß-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

Notch signaling in developmental and tumor angiogenesis.

The discovery that Notch, a key regulator of cell fate determination, is functional in the vasculature has greatly improved our understanding of differentiation and specialization of vessels. Notch signaling has been proven to be critical for arterial specification, sprouting angiogenesis, and vessel maturation. In newly forming vascular sprouts, Notch promotes the distinction between the leading "tip" endothelial cell and the growing "stalk" cell, the endothelial cells that eventually form a new capillary. Notch signaling has also been implicated in vessel stability by regulating vascular mural cell function. More recently, macrophages carrying an activated Notch have been implicated in shaping the course of new sprout formation. Tumor vessels abide by similar principles and use Notch signaling in similar ways. An exciting discovery, made by several researchers, shows that blocking Notch function in tumor vasculature provides a means by which to suppress tumor growth. The authors discuss the developmental and physiological role of Notch in the vasculature and apply this knowledge to an overview of how Notch targeting in the tumor environment can affect tumor angiogenesis and growth.

CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration.

Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b (P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/- cells failed to differentiate when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases (HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression, a requirement for myoblasts to exit the cell cycle and transit to myotubes.

Cross-talk between calcineurin/NFAT and Jak/STAT signalling induces cardioprotective alphaB-crystallin gene expression in response to hypertrophic stimuli.

Among the stress proteins that are up-regulated in the heart due to imposed biomechanical stress, alphaB-crystallin (CryAB) is the most abundant and pivotal in rendering protection against stress-induced cell damage. Cardiomyocyte-specific expression of the CryAB gene was shown to be dependent upon an intact alphaBE4 cis-element located in the CryAB enhancer. To date, there is no evidence on the identity of regulatory proteins and associated signalling molecules that control CryAB expression in cardiomyocytes. In this study, we define a mechanism by which the calcineurin/NFAT and Jak/STAT pathways regulate CryAB gene expression in response to a hypertrophic agonist endothelin-1 (En-1), in hypertrophic hearts of mice with pressure overload (TAC) and in heart-targeted calcineurin over-expressing mice (MHC-CnA). We observed that in response to various hypertrophic stimuli the transcription factors NFAT, Nished and STAT3 form a dynamic ternary complex and interact with the alphaBE4 promoter element of the CryAB gene. Both dominant negative NFAT and AG490, an inhibitor of the Jak2 phosphorylation, inhibited CryAB gene transcription in transient transfection assays. AG490 was also effective in blocking the nuclear translocation of NFAT and STAT3 in cardiomyocytes treated with En-1. We observed a marked increase in CryAB gene expression in MHC-CnA mouse hearts accompanied with increased phosphorylation of STAT3. We conclude that hypertrophy-dependent CryAB gene expression can be attributed to a functional linkage between the Jak/STAT and calcineurin/NFAT signalling pathways, each of which are otherwise known to be involved independently in the deleterious outcome in cardiac hypertrophy.

Repression of the cardiac myosin light chain-2 gene in skeletal muscle requires site-specific association of antithetic regulator, Nished, and HDACs.

The transcriptional activation mechanisms that regulate tissue-specific expression of cardiac muscle genes have been extensively investigated, but little is known of the regulatory events involved in repression of cardiac-specific genes in non-cardiac cells. We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac-Specific Sequence (CSS), in myosin light chain-2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively. Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co-repressor. Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose-dependent manner. Co-transfection studies in primary muscle cells in culture and in Nished expressing stable skeletal muscle cell line demonstrate that Nished down-regulates the cardiac MLC2 gene expression when its association is restricted to CSS alone. Chromatin immunoprecipitation data suggest that the CSS-mediated repression of cardiac MLC2v gene in skeletal muscle cells excludes the participation of the positive element IRE despite the presence of an identical Nished binding site. Taken together, it appears that the negative control of MLC2v transcription is based on a dual mode of regulations, one that affords inaccessibility of IRE to Nished and second that promotes the formation of the transcription repression complex at the inhibitory CSS site to silence the cardiac gene in skeletal muscle cell.

Immune complex deposition in adult male Sprague-Dawley rats chronically immunized with GnRH.

PROBLEM: This study was undertaken to evaluate whether the anti-GnRH antibodies and immune complexes (IC) generated by immunization with GnRH-TT cause cellular damage within the animal. METHOD OF STUDY: Chronic immunization of rats with GnRH-TT injected i.m. was followed by tissue/organ analysis for immune complex deposition by immunofluorescence microscopy. Two groups were studied: (1) those immunized throughout the experiment until their ultimate demise, and (2) those given a chance to recover from the effects of chronic immunization before final analysis. RESULTS: GnRH-TT was effective in stopping spermatogenesis, which resumed after withdrawal of the immunogen. Most tissues from chronically immunized animals were not significantly different than controls, however the kidneys of treated animals exhibited a higher accumulation of IC. Despite increased IC deposition, pathologic effects were not detected at the cellular level. CONCLUSIONS: GnRH-TT is an effective immunocontraceptive although the accumulation of glomerular IC represents a potential deleterious side effect.