Tamar Aprahamian

Postnatal recapitulation of embryonic hedgehog pathway in response to skeletal muscle ischemia.

Background—Hedgehog (Hh) proteins are morphogens regulating epithelial–mesenchymal signaling during several crucial processes of embryonic development, including muscle patterning. Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog constitute the repertoire of Hh genes in humans. The activities of all 3 are transduced via the Patched (Ptc1) receptor. Recent observations indicate that exogenous administration of Shh induces angiogenesis. Here, we studied whether the endogenous Hh pathway, in addition to its functions during embryogenesis, plays a physiological role in muscle regeneration after ischemia in adults. Methods and Results—We found that skeletal muscle ischemia induces strong local upregulation of Shh mRNA and protein. In addition, the Ptc1 receptor is activated in interstitial mesenchymal cells within the ischemic area, indicating that these cells respond to Shh and that the Shh pathway is functional. We also found that Shh-responding cells produce vascular endothelial growth factor under ischemic conditions and that systemic treatment with a Shh-blocking antibody inhibits the local angiogenic response and the upregulation of vascular endothelial growth factor. Conclusions—Our study shows that the Hh signaling may be recapitulated postnatally in adult and fully differentiated muscular tissues and has a regulatory role on angiogenesis during muscle regeneration after ischemia. These findings demonstrate a novel biological activity for the Hh pathway with both fundamental and potential therapeutic implications.

Impaired Clearance of Apoptotic Cells Promotes Synergy between Atherogenesis and Autoimmune Disease

To clarify the link between autoimmune disease and hypercholesterolemia, we created the gld.apoE −/− mouse as a model of accelerated atherosclerosis. Atherosclerotic lesion area was significantly increased in gld.apoE −/− mice compared with apoE −/− mice. gld.apoE −/− mice also displayed increases in lymphadenopathy, splenomegaly, and autoantibodies compared with gld mice, and these effects were exacerbated by high cholesterol diet. gld.apoE −/− mice exhibited higher levels of apoptotic cells, yet a reduced frequency of engulfed apoptotic nuclei within macrophages. Infusion of lysophosphatidylcholine, a component of oxidized low density lipoprotein, markedly decreased apoptotic cell clearance in gld mice, indicating that hypercholesterolemia promotes autoimmune disease in this background. These data suggest that defects in apoptotic cell clearance promote synergy between atherosclerotic and autoimmune diseases.

Alveolar macrophage activation and an emphysema-like phenotype in adiponectin deficient mice

Adiponectin is an adipocyte-derived collectin that acts on a wide range of tissues including liver, brain, heart, and vascular endothelium. To date, little is known about the actions of adiponectin in the lung. Herein, we demonstrate that adiponectin is present in lung lining fluid and that adiponectin deficiency leads to increases in proinflammatory mediators and an emphysema-like phenotype in the mouse lung. Alveolar macrophages from adiponectin-deficient mice spontaneously display increased production of tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase (MMP-12) activity. Consistent with these observations, we found that pretreatment of alveolar macrophages with adiponectin leads to TNF-alpha and MMP-12 suppression. Together, our findings show that adiponectin leads to macrophage suppression in the lung and suggest that adiponectin-deficient states may contribute to the pathogenesis of inflammatory lung conditions such as emphysema.

Selective Activation of Peroxisome Proliferator–Activated Receptor (PPAR)α and PPARγ Induces Neoangiogenesis Through a Vascular Endothelial Growth Factor–Dependent Mechanism

OBJECTIVE—Peroxisome proliferator–activated receptors (PPARs) are therapeutic targets for fibrates and thiazolidinediones, which are commonly used to ameliorate hyperlipidemia and hyperglycemia in type 2 diabetes. In this study, we evaluated whether activation of PPARα and PPARγ stimulates neoangiogenesis. RESEARCH DESIGN AND METHODS—We used selective synthetic PPARα and PPARγ agonists and investigated their angiogenic potentials in vitro and in vivo. RESULTS—Activation of PPARα and PPARγ leads to endothelial tube formation in an endothelial/interstitial cell co-culture assay. This effect is associated with increased production of the angiogenic cytokine vascular endothelial growth factor (VEGF). Neovascularization also occurs in vivo, when PPARα and PPARγ agonists are used in the murine corneal angiogenic model. No vascular growth is detectable when PPARα and PPARγ agonists are respectively used in PPARα knockout mice and mice treated with a specific PPARγ inhibitor, demonstrating that this angiogenic response is PPAR mediated. PPARα- and PPARγ-induced angiogenesis is associated with local VEGF production and does not differ in extent and morphology from that induced by VEGF. In addition, PPARα- and PPARγ-induced in vitro and in vivo angiogenesis may be significantly decreased by inhibiting VEGF activity. Finally, in corneas treated with PPARα and PPARγ agonists, there is increased phosphorylation of endothelial nitric oxide synthase and Akt. CONCLUSIONS—These findings demonstrate that PPARα and PPARγ activation stimulates neoangiogenesis through a VEGF-dependent mechanism. Neoangiogenesis is a crucial pathological event in type 2 diabetes. The ability of PPARα and PPARγ agonists to induce neoangiogenesis might have important implications for the clinical and therapeutic management of type 2 diabetes.

Simvastatin Treatment Ameliorates Autoimmune Disease Associated with Accelerated Atherosclerosis in a Murine Lupus Model

Patients with systemic lupus erythematosus develop accelerated atherosclerosis independent of traditional risk factors. The 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors are widely prescribed for hyperlipidemia, but they also exhibit anti-inflammatory actions that appear to be independent of their suppressive actions on plasma cholesterol levels. In this study, we analyzed the effect of the HMG-CoA reductase inhibitor simvastatin on disease manifestations in gld.apoE−/− mice that lack functional Fas ligand and apolipoprotein E and exhibit accelerated atherosclerosis and aggravated lupus-like features. Wild-type, gld, apoE−/−, and gld.apoE−/− mice were maintained on a high cholesterol Western diet and received daily simvastatin (0.125 mg/kg) or saline for 12 wk. Serum cholesterol levels were unaffected by simvastatin treatment, but atherosclerotic lesion area was reduced in both apoE−/− and gld.apoE−/− mice treated with simvastatin. Simvastatin also reduced the lymphadenopathy, renal disease, and proinflammatory cytokine production seen in gld.apoE−/−, but not gld, mice. The immunomodulatory effects in gld.apoE−/− mice were associated with enhanced STAT6 and decreased STAT4 induction in submandibular lymph node cells. Along with reductions in serum TNF-α and IFN-γ levels, there was also an increase in IL-4 and IL-10 transcript levels in lymph nodes. These data indicate that HMG-CoA reductase inhibitors ameliorate atherosclerosis and lupus-like autoimmunity independent of their cholesterol-lowering effects via a shift from a Th1 to a Th2 phenotype in the gld.apoE−/− model. Thus, the anti-inflammatory activities of statins may have utility for the treatment of both autoimmunity and atherosclerosis in patients with systemic lupus erythematosus.

Sonic Hedgehog Regulates Angiogenesis and Myogenesis During Post-Natal Skeletal Muscle Regeneration

Sonic hedgehog (Shh) is a morphogen‐regulating crucial epithelial‐mesenchymal interactions during embryonic development, but its signalling pathway is considered generally silent in post‐natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant up‐regulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signalling pathway has an important regulatory role on injury‐induced angiogenesis, as inhibition of Shh function results in impaired up‐regulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal‐derived factor (SDF)‐1alpha, decreased muscle blood flow and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf‐5 and MyoD, decreases the up‐regulation of insulin‐like growth factor (IGF)‐1 and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post‐natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.

The Peroxisome Proliferator-Activated Receptor γ Agonist Rosiglitazone Ameliorates Murine Lupus by Induction of Adiponectin

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease for which current therapy is suboptimal. SLE is characterized by autoantibody production, with renal disease and premature atherosclerosis being common and severe manifestations causing appreciable morbidity and mortality. Peroxisome proliferator-activated receptor γ (PPARγ) agonists are widely used in the treatment of diabetes mellitus for their insulin-sensitizing properties, but also have immunomodulatory effects. In this report, we show that the PPARγ agonist rosiglitazone reduces autoantibody production, renal disease, and atherosclerosis in mouse models of SLE. The beneficial effect of rosiglitazone on SLE manifestations depends on the induction of adiponectin, because rosiglitazone has no effect on autoantibody production or renal disease in lupus mice that lack adiponectin. In addition, lupus mice that lack adiponectin develop more severe disease than adiponectin-sufficient lupus mice, indicating that endogenous adiponectin is involved in regulating disease activity. Furthermore, administration of exogenous adiponectin ameliorates disease. These experiments suggest that PPARγ agonists may be useful agents for the treatment of SLE. They also demonstrate that induction of adiponectin is a major mechanism underlying the immunomodulatory effects of PPARγ agonists.

IFN Regulatory Factor 5 Is Required for Disease Development in the FcγRIIB−/−Yaa and FcγRIIB−/− Mouse Models of Systemic Lupus Erythematosus

Polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study, we show that IRF5 is absolutely required for disease development in the FcγRIIB−/−Yaa and FcγRIIB−/− lupus models. In contrast to IRF5-sufficient FcγRIIB−/−Yaa mice, IRF5-deficient FcγRIIB−/−Yaa mice do not develop lupus manifestations and have a phenotype comparable to wild-type mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity, as IRF5 heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I IFN, IFN-α, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcγRIIB−/−Yaa mice lacking the type I IFN receptor subunit 1. Unlike the IRF5-deficient and IRF5-heterozygous FcγRIIB−/−Yaa mice, type I IFN receptor subunit 1-deficient FcγRIIB−/−Yaa mice maintained a substantial level of residual disease. Furthermore, in FcγRIIB−/− mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations, indicating that the beneficial effects of IRF5 deficiency in FcγRIIB−/−Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I IFN production.

Ageing is associated with diminished apoptotic cell clearance in vivo

Ageing leads to immune system dysfunction and the accumulation of autoantibodies. Because the rapid phagocytic clearance of apoptotic cells is required to prevent the development of autoimmunity, we examined the relative clearance of apoptotic material in young and aged mice using two independent assays. First, 2‐year‐old mice were found to be impaired in their ability to clear apoptotic keratinocytes following ultraviolet irradiation of the skin. Secondly, peritoneal macrophages exposed to apoptotic Jurkat T cells in vivo displayed diminished phagocytic activity in aged mice compared with 8‐week‐old mice. Consistent with these findings, aged mice exhibited signs of autoimmunity with the appearance of anti‐nuclear antibodies and increased kidney glomerular size as well as complement deposits within the glomeruli. In vitro assays revealed that the pretreatment of macrophages with the serum from aged mice led to a reduction in their ability to phagocytose apoptotic bodies compared with macrophages treated with serum from young mice. These data show that the ageing process is accompanied by a diminished ability to clear apoptotic debris. This accumulation of apoptotic debris could contribute to immune system dysfunction that occurs in aged organisms.

Adiponectin in cardiovascular inflammation and obesity.

Inflammation is widely known to play a key role in the development and progression of cardiovascular diseases. It is becoming increasingly evident that obesity is linked to many proinflammatory and obesity-associated cardiovascular conditions (e.g., metabolic syndrome, acute coronary syndrome, and congestive heart failure). It has been observed that adipokines play an increasingly large role in systemic and local inflammation. Therefore, adipose tissue may have a more important role than previously thought in the pathogenesis of several disease types. This review explores the recently described role of adiponectin as an immunomodulatory factor and how it intersects with the inflammation associated with both cardiovascular and autoimmune pathologies.