Periostin-related progression of different types of experimental pulmonary hypertension: A role for M2 macrophage and FGF-2 signalling.

BACKGROUND AND OBJECTIVE: Remodelling of pulmonary arteries (PA) contributes to the progression of pulmonary hypertension (PH). Periostin, a matricellular protein, has been reported to be involved in the development of PH. We examined the role of periostin in the pathogenesis of PH using different types of experimental PH. METHODS: PH was induced by vascular endothelial growth factor receptor antagonist (Sugen5416) plus hypoxic exposure (SuHx) and venous injection of monocrotaline-pyrrole (MCT-P) in wild-type (WT) and periostin-/- mice. Pulmonary haemodynamics, PA remodelling, expression of chemokines and fibroblast growth factor (FGF)-2, accumulation of macrophages to small PA and the right ventricle (RV) were examined in PH-induced WT and periostin-/- mice. Additionally, the role of periostin in the migration of macrophages, human PA smooth muscle (HPASMCs) and endothelial cells (HPMVECs) was investigated. RESULTS: In PH induced by SuHx and MCT-P, PH and accumulation of M2 macrophage to small PA were attenuated in periostin-/- mice. PA remodelling post-SuHx treatment was also mild in periostin-/- mice compared to WT mice. Expression of macrophage-associated chemokines and FGF-2 in lung tissue, and accumulation of CD68-positive cells in the RV were less in SuHx periostin-/- than in SuHx WT mice. Periostin secretion in HPASMCs and HPMVECs was enhanced by transforming growth factor-ß. Periostin also augmented macrophage, HPASMCs and HPMVECs migration. Separately, serum periostin levels were significantly elevated in patients with PH compared to healthy controls. CONCLUSION: Periostin is involved in the development of different types of experimental PH, and may also contribute to the pathogenesis of human PH.

Smad7 effects on TGF-ß and ErbB2 restrain myofibroblast activation and protect from postinfarction heart failure.

Repair of the infarcted heart requires TGF-ß/Smad3 signaling in cardiac myofibroblasts. However, TGF-ß-driven myofibroblast activation needs to be tightly regulated in order to prevent excessive fibrosis and adverse remodeling that may precipitate heart failure. We hypothesized that induction of the inhibitory Smad, Smad7, may restrain infarct myofibroblast activation, and we examined the molecular mechanisms of Smad7 actions. In a mouse model of nonreperfused infarction, Smad3 activation triggered Smad7 synthesis in α-SMA+ infarct myofibroblasts, but not in α-SMA-PDGFRα+ fibroblasts. Myofibroblast-specific Smad7 loss increased heart failure-related mortality, worsened dysfunction, and accentuated fibrosis in the infarct border zone and in the papillary muscles. Smad7 attenuated myofibroblast activation and reduced synthesis of structural and matricellular extracellular matrix proteins. Smad7 effects on TGF-ß cascades involved deactivation of Smad2/3 and non-Smad pathways, without any effects on TGF-ß receptor activity. Unbiased transcriptomic and proteomic analysis identified receptor tyrosine kinase signaling as a major target of Smad7. Smad7 interacted with ErbB2 in a TGF-ß-independent manner and restrained ErbB1/ErbB2 activation, suppressing fibroblast expression of fibrogenic proteases, integrins, and CD44. Smad7 induction in myofibroblasts serves as an endogenous TGF-ß-induced negative feedback mechanism that inhibits postinfarction fibrosis by restraining Smad-dependent and Smad-independent TGF-ß responses, and by suppressing TGF-ß-independent fibrogenic actions of ErbB2.

The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition.

Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1-/- mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1-/- cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.

miR-145 transgenic mice develop cardiopulmonary complications leading to postnatal death.

BACKGROUND: Both downregulation and elevation of microRNA miR-145 has been linked to an array of cardiopulmonary phenotypes, and a host of studies suggest that it is an important contributor in governing the differentiation of cardiac and vascular smooth muscle cell types. METHODS AND RESULTS: To better understand the role of elevated miR-145 in utero within the cardiopulmonary system, we utilized a transgene to overexpress miR-145 embryonically in mice and examined the consequences of this lineage-restricted enhanced expression. Overexpression of miR-145 has detrimental effects that manifest after birth as overexpressor mice are unable to survive beyond postnatal day 18. The miR-145 expressing mice exhibit respiratory distress and fail to thrive. Gross analysis revealed an enlarged right ventricle, and pulmonary dysplasia with vascular hypertrophy. Single cell sequencing of RNA derived from lungs of control and miR-145 transgenic mice demonstrated that miR-145 overexpression had global effects on the lung with an increase in immune cells and evidence of leukocyte extravasation associated with vascular inflammation. CONCLUSIONS: These data provide novel findings that demonstrate a pathological role for miR-145 in the cardiopulmonary system that extends beyond its normal function in governing smooth muscle differentiation.

Altered sacral neural crest development in Pax3 spina bifida mutants underlies deficits of bladder innervation and function.

Mouse models of Spina bifida (SB) have been instrumental for identifying genes, developmental processes, and environmental factors that influence neurulation and neural tube closure. Beyond the prominent neural tube defects, other aspects of the nervous system can be affected in SB with significant changes in essential bodily functions such as urination. SB patients frequently experience bladder dysfunction and SB fetuses exhibit reduced density of bladder nerves and smooth muscle although the developmental origins of these deficits have not been determined. The Pax3 Splotch-delayed (Pax3Sp-d) mouse model of SB is one of a very few mouse SB models that survives to late stages of gestation. Through analysis of Pax3Sp-d mutants we sought to define how altered bladder innervation in SB might arise by tracing sacral neural crest (NC) development, pelvic ganglia neuronal differentiation, and assessing bladder nerve fiber density. In Pax3Sp-d/Sp-d fetal mice we observed delayed migration of Sox10+ NC-derived progenitors (NCPs), deficient pelvic ganglia neurogenesis, and reduced density of bladder wall innervation. We further combined NC-specific deletion of Pax3 with the constitutive Pax3Sp-d allele in an effort to generate viable Pax3 mutants to examine later stages of bladder innervation and postnatal bladder function. Neural crest specific deletion of a Pax3 flox allele, using a Sox10-cre driver, in combination with a constitutive Pax3Sp-d mutation produced postnatal viable offspring that exhibited altered bladder function as well as reduced bladder wall innervation and altered connectivity between accessory ganglia at the bladder neck. Combined, the results show that Pax3 plays critical roles within sacral NC that are essential for initiation of neurogenesis and differentiation of autonomic neurons within pelvic ganglia.

Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis.

Heart failure due to high blood pressure or ischemic injury remains a major problem for millions of patients worldwide. Despite enormous advances in deciphering the molecular mechanisms underlying heart failure progression, the cell-type specific adaptations and especially intercellular signaling remain poorly understood. Cardiac fibroblasts express high levels of cardiogenic transcription factors such as GATA-4 and GATA-6, but their role in fibroblasts during stress is not known. Here, we show that fibroblast GATA-4 and GATA-6 promote adaptive remodeling in pressure overload induced cardiac hypertrophy. Using a mouse model with specific single or double deletion of Gata4 and Gata6 in stress activated fibroblasts, we found a reduced myocardial capillarization in mice with Gata4/6 double deletion following pressure overload, while single deletion of Gata4 or Gata6 had no effect. Importantly, we confirmed the reduced angiogenic response using an in vitro co-culture system with Gata4/6 deleted cardiac fibroblasts and endothelial cells. A comprehensive RNA-sequencing analysis revealed an upregulation of anti-angiogenic genes upon Gata4/6 deletion in fibroblasts, and siRNA mediated downregulation of these genes restored endothelial cell growth. In conclusion, we identified a novel role for the cardiogenic transcription factors GATA-4 and GATA-6 in heart fibroblasts, where both proteins act in concert to promote myocardial capillarization and heart function by directing intercellular crosstalk.

Transforming Growth Factor-induced Protein Promotes NF-κB-mediated Angiogenesis during Postnatal Lung Development.

Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-κB promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-κB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-κB signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis. NF-κB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-ß-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PECs. Proteomic analysis identified TGFBI as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NF-κB via αvß3 integrins. RNA sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PECs. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally regulated protein that promotes NF-κB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

Cracd Marks the First Wave of Meiosis during Spermatogenesis and Is Mis-Expressed in Azoospermia Mice.

Testicular development starts in utero and maturation continues postnatally, requiring a cascade of gene activation and differentiation into different cell types, with each cell type having its own specific function. As we had previously reported that the Capping protein inhibiting regulator of actin (Cracd) gene was expressed in the adult mouse testis, herein we examine when and where the ß-catenin associated Cracd is initially expressed during postnatal testis development. Significantly, Cracd mRNA is present in both the immature postnatal and adult testis in round spermatid cells, with highest level of expression occurring during the first wave of meiosis and spermatogenesis. In the juvenile testes, Cracd is initially expressed within the innermost region but as maturation occurs, Cracd mRNA switches to a more peripheral location. Thereafter, Cracd is downregulated to maintenance levels in the haploid male germ cell lineage. As Cracd mRNA was expressed within developing round spermatids, we tested its effectiveness as a biomarker of non-obstructive azoospermia using transgenic knockout mice models. Meaningfully, Cracd expression was absent in Deleted in azoospermia like (Dazl) null testis, which exhibit a dramatic germ cell loss. Moreover, Cracd was abnormally regulated and ectopically mis-expressed in Polypyrimidine tract binding protein-2 (Ptbp2) conditional germ cell restricted knockout testis, which exhibit a block during spermatid differentiation and a reduction in the number of late stage spermatocytes coincident with reduced ß-catenin expression. Combined, these data suggest that Cracd is a useful first wave of spermatogenesis biomarker of azoospermia phenotypes, even prior to an overt phenotype being evident.

Generation of transgenic mice that conditionally express microRNA miR-145.

MicroRNAs are modulators of cellular phenotypes and their functions contribute to development, homeostasis, and disease. miR-145 is a conserved microRNA that has been implicated in regulating an array of phenotypes. These include supporting smooth muscle differentiation, repression of stem cell pluripotency, and inhibition of tumor growth and metastasis. Previously, our lab demonstrated that miR-145 acts to suppress cardiac fibrosis through inhibition of the TGF-ß signaling pathway. The range of effects that miR-145 has on different cell types makes it an attractive microRNA for further study. Here we describe the generation of transgenic mice that conditionally express miR-145 through Cre recombinase-mediated activation. Characterization of individual founder lines indicates that overexpression of miR-145 in the developing cardiovascular system has detrimental effects, with three independent miR-145 transgenic lines exhibiting Cre-dependent lethality. Expression analysis demonstrates that the transgene is robustly expressed and our analysis reveals a novel downstream target of miR-145, Tnnt2. The miR-145 transgenic mice represent a valuable tool to understand the role of miR-145 in diverse cell types and to address its potential as a therapeutic mediator for the treatment of disease.

Cross-Talk between Transforming Growth Factor-ß and Periostin Can Be Targeted for Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized as progressive and irreversible fibrosis in the interstitium of lung tissues. There is still an unmet need to develop a novel therapeutic drug for IPF. We have previously demonstrated that periostin, a matricellular protein, plays an important role in the pathogenesis of pulmonary fibrosis. However, the underlying mechanism of how periostin causes pulmonary fibrosis remains unclear. In this study, we sought to learn whether the cross-talk between TGF-ß (transforming growth factor-ß), a central mediator in pulmonary fibrosis, and periostin in lung fibroblasts leads to generation of pulmonary fibrosis and whether inhibitors for integrin αVß3, a periostin receptor, can block pulmonary fibrosis in model mice and the TGF-ß signals in fibroblasts from patients with IPF. We found that cross-talk exists between TGF-ß and periostin signals via αVß3/ß5 converging into Smad3. This cross-talk is necessary for the expression of TGF-ß downstream effector molecules important for pulmonary fibrosis. Moreover, we identified several potent integrin low-molecular-weight inhibitors capable of blocking cross-talk with TGF-ß signaling. One of the compounds, CP4715, attenuated bleomycin-induced pulmonary fibrosis in vivo in mice and the TGF-ß signals in vitro in fibroblasts from patients with IPF. These results suggest that the cross-talk between TGF-ß and periostin can be targeted for pulmonary fibrosis and that CP4715 can be a potential therapeutic agent to block this cross-talk.

Protective Effects of Activated Myofibroblasts in the Pressure-Overloaded Myocardium Are Mediated Through Smad-Dependent Activation of a Matrix-Preserving Program.

RATIONALE: The heart contains abundant interstitial and perivascular fibroblasts. Traditional views suggest that, under conditions of mechanical stress, cytokines, growth factors, and neurohumoral mediators stimulate fibroblast activation, inducing ECM (extracellular matrix) protein synthesis and promoting fibrosis and diastolic dysfunction. Members of the TGF (transforming growth factor)-ß family are upregulated and activated in the remodeling myocardium and modulate phenotype and function of all myocardial cell types through activation of intracellular effector molecules, the Smads (small mothers against decapentaplegic), and through Smad-independent pathways. OBJECTIVES: To examine the role of fibroblast-specific TGF-ß/Smad3 signaling in the remodeling pressure-overloaded myocardium. METHODS AND RESULTS: We examined the effects of cell-specific Smad3 loss in activated periostin-expressing myofibroblasts using a mouse model of cardiac pressure overload, induced through transverse aortic constriction. Surprisingly, FS3KO (myofibroblast-specific Smad3 knockout) mice exhibited accelerated systolic dysfunction after pressure overload, evidenced by an early 40% reduction in ejection fraction after 7 days of transverse aortic constriction. Accelerated systolic dysfunction in pressure-overloaded FS3KO mice was associated with accentuated matrix degradation and generation of collagen-derived matrikines, accompanied by cardiomyocyte myofibrillar loss and apoptosis, and by enhanced macrophage-driven inflammation. In vitro, TGF-ß1, TGF-ß2, and TGF-ß3 stimulated a Smad3-dependent matrix-preserving phenotype in cardiac fibroblasts, suppressing MMP (matrix metalloproteinase)-3 and MMP-8 synthesis and inducing TIMP (tissue inhibitor of metalloproteinases)-1. In vivo, administration of an MMP-8 inhibitor attenuated early systolic dysfunction in pressure-overloaded FS3KO mice, suggesting that the protective effects of activated cardiac myofibroblasts in the pressure-overloaded myocardium are, at least in part, because of suppression of MMPs and activation of a matrix-preserving program. MMP-8 stimulation induces a proinflammatory phenotype in isolated macrophages. CONCLUSIONS: In the pressure-overloaded myocardium, TGF-ß/Smad3-activated cardiac fibroblasts play an important protective role, preserving the ECM network, suppressing macrophage-driven inflammation, and attenuating cardiomyocyte injury. The protective actions of the myofibroblasts are mediated, at least in part, through Smad-dependent suppression of matrix-degrading proteases.

Myofibroblast ß2 adrenergic signaling amplifies cardiac hypertrophy in mice.

Abnormal ß-adrenergic signaling plays a central role in human heart failure. In mice, chronic ß-adrenergic receptor (ßAR) stimulation elicits cardiac hypertrophy. It has been reported that cultured cardiac fibroblasts express ßAR; however, the functional in vivo requirement of ßAR signaling in cardiac fibroblasts during the development of cardiac hypertrophy remains elusive. ß2AR null mice exhibited attenuated hypertrophic responses to chronic ßAR stimulation upon continuous infusion of an agonist, isoprenaline (ISO), compared to those in wildtype controls, suggesting that ß2AR activation in the heart induces pro-hypertrophic effects in mice. Since ß2AR signaling is protective in cardiomyocytes, we focused on ß2AR signaling in cardiac myofibroblasts. To determine whether ß2AR signaling in myofibroblasts affects cardiac hypertrophy, we generated myofibroblast-specific transgenic mice (TG) with the catalytic subunit of protein kinase A (PKAcα) using Cre-loxP system. Myofibroblast-specific PKAcα overexpression resulted in enhanced heart weight normalized to body weight ratio, associated with an enlargement of cardiomyocytes at 12 weeks of age, indicating that myofibroblast-specific activation of PKA mediates cardiac hypertrophy in mice. Neonatal rat cardiomyocytes stimulated with conditioned media from TG cardiac fibroblasts likewise exhibited significantly more growth than those from controls. Thus, ß2AR signaling in myofibroblasts plays a substantial role in ISO-induced cardiac hypertrophy, possibly due to a paracrine effect. ß2AR signaling in cardiac myofibroblasts may represent a promising target for development of novel therapies for cardiac hypertrophy.

Sca-1+ cardiac fibroblasts promote development of heart failure.

The causative effect of GM-CSF produced by cardiac fibroblasts to development of heart failure has not been shown. We identified the pathological GM-CSF-producing cardiac fibroblast subset and the specific deletion of IL-17A signaling to these cells attenuated cardiac inflammation and heart failure. We describe here the CD45- CD31- CD29+ mEF-SK4+ PDGFRα+ Sca-1+ periostin+ (Sca-1+ ) cardiac fibroblast subset as the main GM-CSF producer in both experimental autoimmune myocarditis and myocardial infarction mouse models. Specific ablation of IL-17A signaling to Sca-1+ periostin+ cardiac fibroblasts (PostnCre Il17rafl/fl ) protected mice from post-infarct heart failure and death. Moreover, PostnCre Il17rafl/fl mice had significantly fewer GM-CSF-producing Sca-1+ cardiac fibroblasts and inflammatory Ly6Chi monocytes in the heart. Sca-1+ cardiac fibroblasts were not only potent GM-CSF producers, but also exhibited plasticity and switched their cytokine production profiles depending on local microenvironments. Moreover, we also found GM-CSF-positive cardiac fibroblasts in cardiac biopsy samples from heart failure patients of myocarditis or ischemic origin. Thus, this is the first identification of a pathological GM-CSF-producing cardiac fibroblast subset in human and mice hearts with myocarditis and ischemic cardiomyopathy. Sca-1+ cardiac fibroblasts direct the type of immune cells infiltrating the heart during cardiac inflammation and drive the development of heart failure.

Loss of periostin ameliorates adipose tissue inflammation and fibrosis in vivo.

Recent evidence suggests that the accumulation of macrophages as a result of obesity-induced adipose tissue hypoxia is crucial for the regulation of tissue fibrosis, but the molecular mechanisms underlying adipose tissue fibrosis are still unknown. In this study, we revealed that periostin (Postn) is produced at extraordinary levels by adipose tissue after feeding with a high-fat diet (HFD). Postn was secreted at least from macrophages in visceral adipose tissue during the development of obesity, possibly due to hypoxia. Postn-/- mice had lower levels of crown-like structure formation and fibrosis in adipose tissue and were protected from liver steatosis. These mice also showed amelioration in systemic insulin resistance compared with HFD-fed WT littermates. Mice deficient in Postn in their hematopoietic compartment also had lower levels of inflammation in adipose tissue, in parallel with a reduction in ectopic lipid accumulation compared with the controls. Our data indicated that the regulation of Postn in visceral fat could be beneficial for the maintenance of healthy adipose tissue in obesity.

Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis.

Emerging evidence demonstrates that megakaryocytes (MK) play key roles in regulating skeletal homeostasis and hematopoiesis. To test if the loss of MK negatively impacts osteoblastogenesis and hematopoiesis, we generated conditional knockout mice where Mpl, the receptor for the main MK growth factor, thrombopoietin, was deleted specifically in MK (Mplf/f;PF4cre). Unexpectedly, at 12 weeks of age, these mice exhibited a 10-fold increase in platelets, a significant expansion of hematopoietic/mesenchymal precursors, and a remarkable 20-fold increase in femoral midshaft bone volume. We then investigated whether MK support hematopoietic stem cell (HSC) function through the interaction of MK with osteoblasts (OB). LSK cells (Lin-Sca1+CD117+, enriched HSC population) were co-cultured with OB+MK for 1 week (1wk OB+MK+LSK) or OB alone (1wk OB+LSK). A significant increase in colony-forming units was observed with cells from 1wk OB+MK cultures. Competitive repopulation studies demonstrated significantly higher engraftment in mice transplanted with cells from 1wk OB+MK+LSK cultures compared to 1wk OB+LSK or LSK cultured alone for 1 week. Furthermore, single-cell expression analysis of OB cultured±MK revealed adiponectin as the most significantly upregulated MK-induced gene, which is required for optimal long-term hematopoietic reconstitution. Understanding the interactions between MK, OB, and HSC can inform the development of novel treatments to enhance both HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.

Chemopreventative celecoxib fails to prevent schwannoma formation or sensorineural hearing loss in genetically engineered murine model of neurofibromatosis type 2.

Mutations in the tumor suppressor gene NF2 lead to Neurofibromatosis type 2 (NF2), a tumor predisposition syndrome characterized by the development of schwannomas, including bilateral vestibular schwannomas with complete penetrance. Recent work has implicated the importance of COX-2 in schwannoma growth. Using a genetically engineered murine model of NF2, we demonstrate that selective inhibition of COX-2 with celecoxib fails to prevent the spontaneous development of schwannomas or sensorineural hearing loss in vivo, despite elevated expression levels of COX-2 in Nf2-deficient tumor tissue. These results suggest that COX-2 is nonessential to schwannomagenesis and that the proposed tumor suppressive effects of NSAIDs on schwannomas may occur through COX-2 independent mechanisms.

Constitutive overexpression of periostin delays wound healing in mouse skin.

Periostin is a matricellular protein involved in development, maintenance, and regulation of tissues and organs via by binding to cell surface integrin receptors. Pathologically, periostin plays an important role in the process of wound healing: as a deficiency of the Postn gene delays wound closure and periostin is consistently up-regulated in response to injury and skin diseases. However, the functional role of elevated periostin in the process of wound healing has not been tested. In this study, we generated Postn-transgenic mice under the control of the CAG promoter/enhancer to investigate the effects of constitutive overexpression of full length periostin during its pathophysiological roles. Transgenic mice showed significant overexpression of periostin in skin, lung, and heart, but no morphological changes were observed. However, when these transgenic mice were injured, periostin overexpression delayed the closure of excisional wounds. Expression of IL-1ß and TNFα, pro-inflammatory cytokines important for wound healing, was significantly decreased in the transgenic mice, prior to delayed healing. Infiltration of neutrophils and macrophages, the main sources of IL-1ß and TNFα, was also down-regulated in the transgenic wound sites. From these data, we conclude that enforced expression of periostin delays wound closure due to reduced infiltration of neutrophils and macrophages followed by down-regulation of IL-1ß and TNFα expression. This suggests that regulated spatiotemporal expression of periostin is important for efficient wound healing and that constitutive periostin overexpression interrupts the normal process of wound closure.

Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin.

Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.

Proteomic profiling of TGFBI-null mouse corneas reveals only minor changes in matrix composition supportive of TGFBI knockdown as therapy against TGFBI-linked corneal dystrophies.

TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI, and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wild-type and TGFBI-/- mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage.

Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium.

BACKGROUND: Transforming growth factor-ßs regulate a wide range of cellular responses by activating Smad-dependent and Smad-independent cascades. In the infarcted heart, Smad3 signaling is activated in both cardiomyocytes and interstitial cells. We hypothesized that cell-specific actions of Smad3 regulate repair and remodeling in the infarcted myocardium. METHODS: To dissect cell-specific Smad3 actions in myocardial infarction, we generated mice with Smad3 loss in activated fibroblasts or cardiomyocytes. Cardiac function was assessed after reperfused or nonreperfused infarction using echocardiography. The effects of cell-specific Smad3 loss on the infarcted heart were studied using histological studies, assessment of protein, and gene expression levels. In vitro, we studied Smad-dependent and Smad-independent actions in isolated cardiac fibroblasts. RESULTS: Mice with fibroblast-specific Smad3 loss had accentuated adverse remodeling after reperfused infarction and exhibited an increased incidence of late rupture after nonreperfused infarction. The consequences of fibroblast-specific Smad3 loss were not a result of effects on acute infarct size but were associated with unrestrained fibroblast proliferation, impaired scar remodeling, reduced fibroblast-derived collagen synthesis, and perturbed alignment of myofibroblast arrays in the infarct. Polarized light microscopy in Sirius red-stained sections demonstrated that the changes in fibroblast morphology were associated with perturbed organization of the collagenous matrix in the infarcted area. In contrast, α-smooth muscle actin expression by infarct myofibroblasts was not affected by Smad3 loss. Smad3 critically regulated fibroblast function, activating integrin-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2) expression. Smad3 loss in cardiomyocytes attenuated remodeling and dysfunction after infarction. Cardiomyocyte-specific Smad3 loss did not affect acute infarct size but was associated with attenuated cardiomyocyte apoptosis in the remodeling myocardium, accompanied by decreased myocardial NOX-2 levels, reduced nitrosative stress, and lower matrix metalloproteinase-2 expression. CONCLUSIONS: In healing myocardial infarction, myofibroblast- and cardiomyocyte-specific activation of Smad3 has contrasting functional outcomes that may involve activation of an integrin/reactive oxygen axis.