Critical role of the BAF chromatin remodeling complex during murine neural crest development.

The BAF complex plays an important role in the development of a wide range of tissues by modulating gene expression programs at the chromatin level. However, its role in neural crest development has remained unclear. To determine the role of the BAF complex, we deleted BAF155/BAF170, the core subunits required for the assembly, stability, and functions of the BAF complex in neural crest cells (NCCs). Neural crest-specific deletion of BAF155/BAF170 leads to embryonic lethality due to a wide range of developmental defects including craniofacial, pharyngeal arch artery, and OFT defects. RNAseq and transcription factor enrichment analysis revealed that the BAF complex modulates the expression of multiple signaling pathway genes including Hippo and Notch, essential for the migration, proliferation, and differentiation of the NCCs. Furthermore, we demonstrated that the BAF complex is essential for the Brg1-Yap-Tead-dependent transcription of target genes in NCCs. Together, our results demonstrate an important role of the BAF complex in modulating the gene regulatory network essential for neural crest development.

YAP/TAZ deficiency reprograms macrophage phenotype and improves infarct healing and cardiac function after myocardial infarction.

Adverse cardiac remodeling after myocardial infarction (MI) causes structural and functional changes in the heart leading to heart failure. The initial post-MI pro-inflammatory response followed by reparative or anti-inflammatory response is essential for minimizing the myocardial damage, healing, and scar formation. Bone marrow-derived macrophages (BMDMs) are recruited to the injured myocardium and are essential for cardiac repair as they can adopt both pro-inflammatory or reparative phenotypes to modulate inflammatory and reparative responses, respectively. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the key mediators of the Hippo signaling pathway and are essential for cardiac regeneration and repair. However, their functions in macrophage polarization and post-MI inflammation, remodeling, and healing are not well established. Here, we demonstrate that expression of YAP and TAZ is increased in macrophages undergoing pro-inflammatory or reparative phenotype changes. Genetic deletion of YAP/TAZ leads to impaired pro-inflammatory and enhanced reparative response. Consistently, YAP activation enhanced pro-inflammatory and impaired reparative response. We show that YAP/TAZ promote pro-inflammatory response by increasing interleukin 6 (IL6) expression and impede reparative response by decreasing Arginase-I (Arg1) expression through interaction with the histone deacetylase 3 (HDAC3)-nuclear receptor corepressor 1 (NCoR1) repressor complex. These changes in macrophages polarization due to YAP/TAZ deletion results in reduced fibrosis, hypertrophy, and increased angiogenesis, leading to improved cardiac function after MI. Also, YAP activation augmented MI-induced cardiac fibrosis and remodeling. In summary, we identify YAP/TAZ as important regulators of macrophage-mediated pro-inflammatory or reparative responses post-MI.

Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo.

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/ß-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha (PdgfRα) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/ß-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα.

Deficiency in the secreted protein Semaphorin3d causes abnormal parathyroid development in mice.

Primary hyperparathyroidism (PHPT) is a common endocrinopathy characterized by hypercalcemia and elevated levels of parathyroid hormone. The primary cause of PHPT is a benign overgrowth of parathyroid tissue causing excessive secretion of parathyroid hormone. However, the molecular etiology of PHPT is incompletely defined. Here, we demonstrate that semaphorin3d (Sema3d), a secreted glycoprotein, is expressed in the developing parathyroid gland in mice. We also observed that genetic deletion of Sema3d leads to parathyroid hyperplasia, causing PHPT. In vivo and in vitro experiments using histology, immunohistochemistry, biochemical, RT-qPCR, and immunoblotting assays revealed that Sema3d inhibits parathyroid cell proliferation by decreasing the epidermal growth factor receptor (EGFR)/Erb-B2 receptor tyrosine kinase (ERBB) signaling pathway. We further demonstrate that EGFR signaling is elevated in Sema3d-/- parathyroid glands and that pharmacological inhibition of EGFR signaling can partially rescue the parathyroid hyperplasia phenotype. We propose that because Sema3d is a secreted protein, it may be possible to use recombinant Sema3d or derived peptides to inhibit parathyroid cell proliferation causing hyperplasia and hyperparathyroidism. Collectively, these findings identify Sema3d as a negative regulator of parathyroid growth.

Plexin D1 determines body fat distribution by regulating the type V collagen microenvironment in visceral adipose tissue.

Genome-wide association studies have implicated PLEXIN D1 (PLXND1) in body fat distribution and type 2 diabetes. However, a role for PLXND1 in regional adiposity and insulin resistance is unknown. Here we use in vivo imaging and genetic analysis in zebrafish to show that Plxnd1 regulates body fat distribution and insulin sensitivity. Plxnd1 deficiency in zebrafish induced hyperplastic morphology in visceral adipose tissue (VAT) and reduced lipid storage. In contrast, subcutaneous adipose tissue (SAT) growth and morphology were unaffected, resulting in altered body fat distribution and a reduced VAT:SAT ratio in zebrafish. A VAT-specific role for Plxnd1 appeared conserved in humans, as PLXND1 mRNA was positively associated with hypertrophic morphology in VAT, but not SAT. In zebrafish plxnd1 mutants, the effect on VAT morphology and body fat distribution was dependent on induction of the extracellular matrix protein collagen type V alpha 1 (col5a1). Furthermore, after high-fat feeding, zebrafish plxnd1 mutant VAT was resistant to expansion, and excess lipid was disproportionately deposited in SAT, leading to an even greater exacerbation of altered body fat distribution. Plxnd1-deficient zebrafish were protected from high-fat-diet-induced insulin resistance, and human VAT PLXND1 mRNA was positively associated with type 2 diabetes, suggesting a conserved role for PLXND1 in insulin sensitivity. Together, our findings identify Plxnd1 as a novel regulator of VAT growth, body fat distribution, and insulin sensitivity in both zebrafish and humans.

From basic mechanisms to clinical applications in heart protection, new players in cardiovascular diseases and cardiac theranostics: meeting report from the third international symposium on "New frontiers in cardiovascular research".

In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients' cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia-reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients' outcome.

Semaphorin 3d and semaphorin 3e direct endothelial motility through distinct molecular signaling pathways.

Class 3 semaphorins were initially described as axonal growth cone guidance molecules that signal through plexin and neuropilin coreceptors and since then have been established to be regulators of vascular development. Semaphorin 3e (Sema3e) has been shown previously to repel endothelial cells and is the only class 3 semaphorin known to be capable of signaling via a plexin receptor without a neuropilin coreceptor. Sema3e signals through plexin D1 (Plxnd1) to regulate vascular patterning by modulating the cytoskeleton and focal adhesion structures. We showed recently that semaphorin 3d (Sema3d) mediates endothelial cell repulsion and pulmonary vein patterning during embryogenesis. Here we show that Sema3d and Sema3e affect human umbilical vein endothelial cells similarly but through distinct molecular signaling pathways. Time-lapse imaging studies show that both Sema3d and Sema3e can inhibit cell motility and migration, and tube formation assays indicate that both can impede tubulogenesis. Endothelial cells incubated with either Sema3d or Sema3e demonstrate a loss of actin stress fibers and focal adhesions. However, the addition of neuropilin 1 (Nrp1)-blocking antibody or siRNA knockdown of Nrp1 inhibits Sema3d-mediated, but not Sema3e-mediated, cytoskeletal reorganization, and siRNA knockdown of Nrp1 abrogates Sema3d-mediated, but not Sema3e-mediated, inhibition of tubulogenesis. On the other hand, endothelial cells deficient in Plxnd1 are resistant to endothelial repulsion mediated by Sema3e but not Sema3d. Unlike Sema3e, Sema3d incubation results in phosphorylation of Akt in human umbilical vein endothelial cells, and inhibition of the PI3K/Akt pathway blocks the endothelial guidance and cytoskeletal reorganization functions of Sema3d but not Sema3e.

Murine craniofacial development requires Hdac3-mediated repression of Msx gene expression.

Craniofacial development is characterized by reciprocal interactions between neural crest cells and neighboring cell populations of ectodermal, endodermal and mesodermal origin. Various genetic pathways play critical roles in coordinating the development of cranial structures by modulating the growth, survival and differentiation of neural crest cells. However, the regulation of these pathways, particularly at the epigenomic level, remains poorly understood. Using murine genetics, we show that neural crest cells exhibit a requirement for the class I histone deacetylase Hdac3 during craniofacial development. Mice in which Hdac3 has been conditionally deleted in neural crest demonstrate fully penetrant craniofacial abnormalities, including microcephaly, cleft secondary palate and dental hypoplasia. Consistent with these abnormalities, we observe dysregulation of cell cycle genes and increased apoptosis in neural crest structures in mutant embryos. Known regulators of cell cycle progression and apoptosis in neural crest, including Msx1, Msx2 and Bmp4, are upregulated in Hdac3-deficient cranial mesenchyme. These results suggest that Hdac3 serves as a critical regulator of craniofacial morphogenesis, in part by repressing core apoptotic pathways in cranial neural crest cells.

Lymphatic endothelial progenitors bud from the cardinal vein and intersomitic vessels in mammalian embryos.

The lymphatic vasculature preserves tissue fluid balance by absorbing fluid and macromolecules and transporting them to the blood vessels for circulation. The stepwise process leading to the formation of the mammalian lymphatic vasculature starts by the expression of the gene Prox1 in a subpopulation of blood endothelial cells (BECs) on the cardinal vein (CV) at approximately E9.5. These Prox1-expressing lymphatic endothelial cells (LECs) will exit the CV to form lymph sacs, primitive structures from which the entire lymphatic network is derived. Until now, no conclusive information was available regarding the cellular processes by which these LEC progenitors exit the CV without compromising the vein's integrity. We determined that LECs leave the CV by an active budding mechanism. During this process, LEC progenitors are interconnected by VE-cadherin-expressing junctions. Surprisingly, we also found that Prox1-expressing LEC progenitors were present not only in the CV but also in the intersomitic vessels (ISVs). Furthermore, as LEC progenitors bud from the CV and ISVs into the surrounding mesenchyme, they begin expressing the lymphatic marker podoplanin, migrate away from the CV, and form the lymph sacs. Analyzing this process in Prox1-null embryos revealed that Prox1 activity is necessary for LEC progenitors to exit the CV.

Resolution of defective dorsal aortae patterning in Sema3E-deficient mice occurs via angiogenic remodeling.

BACKGROUND: Neuronal guidance cues influence endothelial cell (EC) behavior to shape the embryonic vascular system. The repulsive neuronal guidance cue, Semaphorin 3E (Sema3E), is critical for creating avascular zones that instruct and subsequently pattern the first embryonic vessels, the paired dorsal aortae (DA). Sema3E(-) (/) (-) embryos develop highly branched plexus-like vessels during vasculogenesis, instead of smooth paired vessels. Unexpectedly, despite these severe DA patterning defects, mutant mice are viable throughout adulthood. RESULTS: Examination of Sema3E(-) (/) (-) mice reveals that the plexus-like DA resolve into single, unbranched vessels between embryonic day (E) E8.25 and E8.75. Although fusion of Sema3E(-) (/) (-) DA occurs slightly earlier than in heterozygotes, the DA are otherwise indistinguishable, suggesting a complete "rescue" in their development. Resolution of the DA null plexuses occurs by remodeling rather than by means of changes in cell proliferation or death. CONCLUSIONS: Normalization of Sema3E(-) (/) (-) DA patterning defects demonstrates resilience of embryonic vascular patterning programs. Additional repulsive guidance cues within the lateral plate mesoderm likely re-establish avascular zones lost in Sema3E(-) (/) (-) embryos and guide resolution of mutant plexus into branchless, parallel aortae. Our observations explain how Sema3E(-) (/) (-) mice survive throughout development and into adulthood, despite severe initial vascular defects.

Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2.

The cardiovascular system generates and responds to mechanical forces. The heartbeat pumps blood through a network of vascular tubes, which adjust their caliber in response to the hemodynamic environment. However, how endothelial cells in the developing vascular system integrate inputs from circulatory forces into signaling pathways to define vessel caliber is poorly understood. Using vertebrate embryos and in vitro-assembled microvascular networks of human endothelial cells as models, flow and genetic manipulations, and custom software, we reveal that Plexin-D1, an endothelial Semaphorin receptor critical for angiogenic guidance, employs its mechanosensing activity to serve as a crucial positive regulator of the Dorsal Aorta's (DA) caliber. We also uncover that the flow-responsive transcription factor KLF2 acts as a paramount mechanosensitive effector of Plexin-D1 that enlarges endothelial cells to widen the vessel. These findings illuminate the molecular and cellular mechanisms orchestrating the interplay between cardiovascular development and hemodynamic forces.

MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development.

The epicardium is a sheet of epithelial cells covering the heart during early cardiac development. In recent years, the epicardium has been identified as an important contributor to cardiovascular development, and epicardium-derived cells have the potential to differentiate into multiple cardiac cell lineages. Some epicardium-derived cells that undergo epithelial-to-mesenchymal transition and delaminate from the surface of the developing heart subsequently invade the myocardium and differentiate into vascular smooth muscle of the developing coronary vasculature. MicroRNAs (miRNAs) have been implicated broadly in tissue patterning and development, including in the heart, but a role in epicardium is unknown. To examine the role of miRNAs during epicardial development, we conditionally deleted the miRNA-processing enzyme Dicer in the proepicardium using Gata5-Cre mice. Epicardial Dicer mutant mice are born in expected Mendelian ratios but die immediately after birth with profound cardiac defects, including impaired coronary vessel development. We found that loss of Dicer leads to impaired epicardial epithelial-to-mesenchymal transition and a reduction in epicardial cell proliferation and differentiation into coronary smooth muscle cells. These results demonstrate a critical role for Dicer, and by implication miRNAs, in murine epicardial development.

New Insights into Hippo/YAP Signaling in Fibrotic Diseases.

Fibrosis results from defective wound healing processes often seen after chronic injury and/or inflammation in a range of organs. Progressive fibrotic events may lead to permanent organ damage/failure. The hallmark of fibrosis is the excessive accumulation of extracellular matrix (ECM), mostly produced by pathological myofibroblasts and myofibroblast-like cells. The Hippo signaling pathway is an evolutionarily conserved kinase cascade, which has been described well for its crucial role in cell proliferation, apoptosis, cell fate decisions, and stem cell self-renewal during development, homeostasis, and tissue regeneration. Recent investigations in clinical and pre-clinical models has shown that the Hippo signaling pathway is linked to the pathophysiology of fibrotic diseases in many organs including the lung, heart, liver, kidney, and skin. In this review, we have summarized recent evidences related to the contribution of the Hippo signaling pathway in the development of organ fibrosis. A better understanding of this pathway will guide us to dissect the pathophysiology of fibrotic disorders and develop effective tissue repair therapies.

Emerging roles of the Hippo signaling pathway in modulating immune response and inflammation-driven tissue repair and remodeling.

Inflammation is an evolutionarily conserved process and part of the body's defense mechanism. Inflammation leads to the activation of immune and non-immune cells that protect the host tissue/organs from injury or intruding pathogens. The Hippo pathway is an evolutionarily conserved kinase cascade with an established role in regulating cell proliferation, survival, and differentiation. It is involved in diverse biological processes, including organ size control and tissue homeostasis. Recent clinical and pre-clinical studies have shown that the Hippo signaling pathway is also associated with injury- and pathogen-induced tissue inflammation and associated immunopathology. In this review, we have summarized the recent findings related to the involvement of the Hippo signaling pathway in modulating the immune response in different acute and chronic inflammatory diseases and its impact on tissue repair and remodeling.

Loss of Yap/Taz in cardiac fibroblasts attenuates adverse remodelling and improves cardiac function.

AIMS: Fibrosis is associated with all forms of adult cardiac diseases including myocardial infarction (MI). In response to MI, the heart undergoes ventricular remodelling that leads to fibrotic scar due to excessive deposition of extracellular matrix mostly produced by myofibroblasts. The structural and mechanical properties of the fibrotic scar are critical determinants of heart function. Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz) are the key effectors of the Hippo signalling pathway and are crucial for cardiomyocyte proliferation during cardiac development and regeneration. However, their role in cardiac fibroblasts, regulating post-MI fibrotic and fibroinflammatory response, is not well established. METHODS AND RESULTS: Using mouse model, we demonstrate that Yap/Taz are activated in cardiac fibroblasts after MI and fibroblasts-specific deletion of Yap/Taz using Col1a2Cre(ER)T mice reduces post-MI fibrotic and fibroinflammatory response and improves cardiac function. Consistently, Yap overexpression elevated post-MI fibrotic response. Gene expression profiling shows significant downregulation of several cytokines involved in post-MI cardiac remodelling. Furthermore, Yap/Taz directly regulate the promoter activity of pro-fibrotic cytokine interleukin-33 (IL33) in cardiac fibroblasts. Blocking of IL33 receptor ST2 using the neutralizing antibody abrogates the Yap-induced pro-fibrotic response in cardiac fibroblasts. We demonstrate that the altered fibroinflammatory programme not only affects the nature of cardiac fibroblasts but also the polarization as well as infiltration of macrophages in the infarcted hearts. Furthermore, we demonstrate that Yap/Taz act downstream of both Wnt and TGFß signalling pathways in regulating cardiac fibroblasts activation and fibroinflammatory response. CONCLUSION: We demonstrate that Yap/Taz play an important role in controlling MI-induced cardiac fibrosis by modulating fibroblasts proliferation, transdifferentiation into myofibroblasts, and fibroinflammatory programme.

The E3 ubiquitin ligase WWP2 regulates pro-fibrogenic monocyte infiltration and activity in heart fibrosis.

Non-ischemic cardiomyopathy (NICM) can cause left ventricular dysfunction through interstitial fibrosis, which corresponds to the failure of cardiac tissue remodeling. Recent evidence implicates monocytes/macrophages in the etiopathology of cardiac fibrosis, but giving their heterogeneity and the antagonizing roles of macrophage subtypes in fibrosis, targeting these cells has been challenging. Here we focus on WWP2, an E3 ubiquitin ligase that acts as a positive genetic regulator of human and murine cardiac fibrosis, and show that myeloid specific deletion of WWP2 reduces cardiac fibrosis in hypertension-induced NICM. By using single cell RNA sequencing analysis of immune cells in the same model, we establish the functional heterogeneity of macrophages and define an early pro-fibrogenic phase of NICM that is driven by Ccl5-expressing Ly6chigh monocytes. Among cardiac macrophage subtypes, WWP2 dysfunction primarily affects Ly6chigh monocytes via modulating Ccl5, and consequentially macrophage infiltration and activation, which contributes to reduced myofibroblast trans-differentiation. WWP2 interacts with transcription factor IRF7, promoting its non-degradative mono-ubiquitination, nuclear translocation and transcriptional activity, leading to upregulation of Ccl5 at transcriptional level. We identify a pro-fibrogenic macrophage subtype in non-ischemic cardiomyopathy, and demonstrate that WWP2 is a key regulator of IRF7-mediated Ccl5/Ly6chigh monocyte axis in heart fibrosis.

Caffeine prevents restenosis and inhibits vascular smooth muscle cell proliferation through the induction of autophagy.

Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.

Distinct enhancers at the Pax3 locus can function redundantly to regulate neural tube and neural crest expressions.

Pax3 is a transcription factor expressed in somitic mesoderm, dorsal neural tube and pre-migratory neural crest during embryonic development. We have previously identified cis-acting enhancer elements within the proximal upstream genomic region of Pax3 that are sufficient to direct functional expression of Pax3 in neural crest. These elements direct expression of a reporter gene to pre-migratory neural crest in transgenic mice, and transgenic expression of a Pax3 cDNA using these elements is sufficient to rescue neural crest development in mice otherwise lacking endogenous Pax3. We show here that deletion of these enhancer sequences by homologous recombination is insufficient to abrogate neural crest expression of Pax3 and results in viable mice. We identify a distinct enhancer in the fourth intron that is also capable of mediating neural crest expression in transgenic mice and zebrafish. Our analysis suggests the existence of functionally redundant neural crest enhancer modules for Pax3.

Gata4 and Gata5 cooperatively regulate cardiac myocyte proliferation in mice.

GATA5 is a member of the zinc finger transcription factor GATA family (GATA1-6) that plays a wide variety of roles in embryonic and adult development. Experiments in multiple model systems have emphasized the importance of the GATA family members 4-6 in the development of the endoderm and mesoderm. Yet despite overlapping expression patterns, there is little evidence of an important role for GATA5 in mammalian cardiac development. We have generated a new Gata5 mutant allele lacking exons 2 and 3 that encodes both zinc finger domains (Gata5(tm)(2)(Eem)), and we show that although Gata5(-/-) mice are viable, Gata4(+/-)5(-/-) mutants die at mid-gestation and exhibit profound cardiovascular defects, including abnormalities of cardiomyocyte proliferation and cardiac chamber maturation. These results demonstrate functional redundancy between Gata4 and Gata5 during cardiac development and implicate Gata5 as a candidate modifier gene for congenital heart disease.