While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here, we report that the deletion of Ptpmt1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection – the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. Ptpmt1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, Ptpmt1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that Ptpmt1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources. Research organism: Mouse

Background: Aortic valve (AV) calcification preferentially occurs on the fibrosa side while the ventricularis side remains relatively unaffected. Here, we tested the hypothesis that side-dependent activation of bone morphogenic protein (BMP) pathway in the endothelium of the ventricularis and fibrosa is associated with human AV calcification. Methods and Results: Human calcified AVs obtained from AV replacement surgeries and non-calcified AVs from heart transplantations were used for immunohistochemical studies. We found SMAD-1/5/8 phosphorylation (a canonical BMP pathway) was higher in the calcified fibrosa than the non-calcified fibrosa while SMAD-2/3 phosphorylation (a canonical TGFβ pathway) did not show any difference. Interestingly, we found that BMP-2/4/6 expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both calcified and non-calcified AV cusps; however, BMP antagonists (crossvienless-2/BMPER and noggin) expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both disease states. Moreover, significant expression of inhibitory SMAD-6 expression was found only in the non-calcified ventricularis endothelium. Conclusions: SMAD-1/5/8 is preferentially activated in the calcified fibrosa endothelium of human AVs and it correlates with low expression of BMP antagonists and inhibitory SMAD6. These results suggest a dominant role of BMP antagonists in the side-dependent calcification of human AVs.

Objective-The adaptive response to vascular injury is the formation of functional collateral vessels to maintain organ integrity. Many of the clinical complications associated with sickle cell disease can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of collateral vessel formation after injury are largely unknown in sickle cell disease. Here, we characterize postischemic neovascularization in sickle cell disease and the role of neutrophils in the production of reactive oxygen species. Approach and Results-We induced hindlimb ischemia by ligation of the femoral artery in Townes SS (sickle cell) mice compared with AA (wild type) mice. Perfusion recovery, ascertained using LASER (light amplification by stimulated emission of radiation) Doppler perfusion imaging, showed significant diminution in collateral vessel formation in SS mice after hindlimb ischemia (76±13% AA versus 34±10% in SS by day 28; P<0.001; n=10 per group). The incidence of amputation (25% versus 5%) and foot necrosis (80% versus 15%) after hindlimb ischemia was significantly increased in the SS mice. Motor function recovery evaluation by the running wheel assay was also impaired in SS mice (36% versus 97% at 28 days post-hindlimb ischemia; P<0.001). This phenotype was associated with persistent and excessive production of reactive oxygen species by neutrophils. Importantly, neutrophil depletion or treatment with the antioxidant N-acetylcysteine reduced oxidative stress and improved functional collateral formation in the SS mice. Conclusions-Our data suggest dysfunctional collateral vessel formation in SS mice after vascular injury and provide a mechanistic basis for the multiple vascular complications of sickle cell disease. Visual Overview-An online visual overview is available for this article.

Diabetics often have poor perfusion in their limbs as a result of peripheral artery disease and an impaired ability to generate collateral vessels. The receptor for advanced glycation end products (RAGE) is one protein that is thought to play a detrimental role in collateral development in diabetics due to increased levels of advanced glycation end products (AGE), one of its ligands, in diabetes. Thus, the aim of this study was to investigate the role of RAGE in both diabetic and non-diabetic settings in a model of collateral formation in mice. Streptozotocin was used to induce diabetes in both wild type and RAGE knockout mice. Increased levels of the AGE, N ϵ -(carboxymethyl) lysine (CML), were confirmed via an ELISA. A hindlimb ischemia model, in which the femoral artery is ligated, was used to drive collateral growth and reperfusion was assessed using laser Doppler perfusion imaging and histological analysis of vessels in the muscle. Both of these measurements showed impaired collateral growth in diabetic compared with wild-type mice as well as improved collateral growth in both diabetic and non-diabetic RAGE knockout mice when compared their wild-type counterparts. Distance on a freely accessed running wheel, used as a measure of perfusion recovery, showed that wild-type diabetic mice had functionally impaired recovery compared with their wild-type counterparts. Immunohistochemistry and immunoblotting showed that HMGB-1 (high-mobility group box 1), another RAGE ligand, was increased in the ischemic leg compared with the non-ischemic leg in all mice. This increase in HMGB-1 may explain improvement in animals lacking RAGE and its subsequent signaling. In conclusion, this study shows that RAGE impairs collateral growth in a diabetic setting and also in a non-diabetic setting. This demonstrates the importance of RAGE and alternate RAGE ligands in the setting of collateral vessel growth.

The dual specificity phosphatase slingshot homolog 1 (SSH1) contributes to actin remodeling by dephosphorylating and activating the actin-severing protein cofilin. The reorganization of the actin cytoskeleton has been implicated in chronic hypertension and the subsequent mechano-adaptive rearrangement of vessel wall components. Therefore, using a novel Ssh1 −/− mouse model, we investigated the potential role of SSH1 in angiotensin II (Ang II)-induced hypertension, and vascular remodeling. We found that loss of SSH1 did not produce overt phenotypic changes and that baseline blood pressures as well as heart rates were comparable between Ssh1 +/+ and Ssh1 −/− mice. Although 14 days of Ang II treatment equally increased systolic blood pressure in both genotypes, histological assessment of aortic samples indicated that medial thickening was exacerbated by the loss of SSH1. Consequently, reverse-transcription quantitative PCR analysis of the transcripts from Ang II-infused animals confirmed increased aortic expression levels of fibronectin, and osteopontin in Ssh1 −/− when compared to wild-type mice. Mechanistically, our data suggest that fibrosis in SSH1-deficient mice occurs by a process that involves aberrant responses to Ang II-induced TGFβ1. Taken together, our work indicates that Ang II-dependent fibrotic gene expression and vascular remodeling, but not the Ang II-induced pressor response, are modulated by SSH1-mediated signaling pathways and SSH1 activity is protective against Ang II-induced remodeling in the vasculature.

Activated lymphocytes promote inflammation and bone destruction in rheumatoid arthritis (RA), making T cells and B cells therapeutic targets. Indeed, pharmacological blockade of CD28 costimulation using CTLA-4Ig (abatacept), approved for amelioration of RA, renders T cells dormant (anergic). CTLA-4Ig also promotes bone accretion in healthy mice; surprisingly, however, this effect is driven exclusively through upregulation of bone formation, rather than anti-inflammatory effects on resorption. In the study presented here, we utilized T cell receptor β gene and Wnt-10b gene knockout mice to investigate the roles of T cells and Wnt-10b in CTLA-4Ig–induced bone anabolism. Ablation of either T cells or Wnt-10b not only abolished CTLA-4Ig–induced bone anabolism but also, paradoxically, suppressed bone formation leading to bone loss. Stalled bone formation was accompanied by bone marrow stromal cell expression of the Wnt pathway inhibitor sclerostin. Our data suggest that an immunoskeletal pivot may promote or suppress bone formation, depending on the net outcome of CTLA-4Ig action directed independently on T cells and osteoblast-linage cells that counter Wnt-10b–induced bone anabolism, by secretion of sclerostin. While CTLA-4Ig action is tipped in favor of bone formation under physiological conditions, pathological immunodeficiency may lead to suppressed bone formation and skeletal damage.

Hemophilia A (HA), a rare X-linked recessive genetic disorder caused by insufficient blood clotting factor VIII, leaves affected individuals susceptible to spontaneous and traumatic hemorrhage. Although males generally exhibit severe symptoms, due to variable X inactivation, females can also be severely impacted. Osteoporosis is a disease of the skeleton predisposing patients to fragility fracture, a cause of significant morbidity and mortality and a common comorbidity in HA. Because the causes of osteoporosis in HA are unclear and in humans confounded by other traditional risk factors for bone loss, in this study, we phenotyped the skeletons of F8 total knockout (F8TKO) mice, an animal model of severe HA. We found that trabecular bone accretion in the axial and appendicular skeletons of male F8TKO mice lagged significantly between 2 and 6 months of age, with more modest cortical bone decline. By contrast, in female mice, diminished bone accretion was mostly limited to the cortical compartment. Interestingly, bone loss was associated with a decline in bone formation in male mice but increased bone resorption in female mice, a possible result of sex steroid insufficiency. In conclusion, our studies reveal a sexual dimorphism in the mechanism driving bone loss in male and female F8TKO mice, preventing attainment of peak bone mass and strength. If validated in humans, therapies aimed at promoting bone formation in males but suppressing bone resorption in females may be indicated to facilitate attainment of peak mass in children with HA to reduce the risk for fracture later in life.

Objective Previous findings from our laboratory demonstrated that neo-vascularization was impaired in osteopontin (OPN) knockout animals. However, the mechanisms responsible for regulation of OPN expression in the setting of ischemia remain undefined. Therefore, we sought to determine if OPN is upregulated in response to ischemia and hypothesized that H2O2 is a critical component of the signaling mechanism by which OPN expression is upregulated in response to ischemia in vivo. Methods and Results To determine if ischemic injury upregulates OPN, we used a murine model of hind limb ischemia. Femoral artery ligation in C57Bl/6 mice significantly increased OPN expression and H2O2 production. Infusion of C57Bl/6 mice with PEG-catalase (10,000 U/kg/day) or the use of transgenic mice with smooth muscle cell specific catalase overexpression blunted ischemia-induced OPN, suggesting ischemia-induced OPN expression is H2O2-dependent. Decreased H2O2-mediated OPN blunted reperfusion and collateral formation in vivo. In contrast, the overexpression of OPN using lentivirus restored neovascularization. Conclusions Scavenging H2O2 blocks ischemia-induced OPN expression, providing evidence that ischemia-induced OPN expression is H2O2-dependent. Decreased OPN expression impaired neo-vascularization, whereas overexpression of OPN increased angiogenesis, supporting our hypothesis that OPN is a critical mediator of post-ischemic neo-vascularization and a potential novel therapeutic target for inducing new vessel growth.

Objectives We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explore whether reducing macrophage intracellular iron levels via pharmacologic suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. Methods and Results To suppress hepcidin, increase expression of the iron exporter ferroportin (FPN), and reduce macrophage intracellular iron, we used a small molecule inhibitor of BMP signaling, LDN 193189 (LDN). LDN (10 mg/kg i.p. bid) was administered to mice and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to Apo E (-/-) mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; p=0.03), reduced oil-red-o positive lipid area by 50% (n=8; p=0.02) and decreased total plaque area by 43% (n=8; p=0.001). LDN suppressed liver hepcidin transcription and increased macrophage FPN, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1 and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting that modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. Conclusion These data suggest that pharmacologic manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

Objective Myeloid lineage cells (MLCs) such as macrophages are known to play a key role in post-ischemic neovascularization. However, the role of MLC-derived reactive oxygen species (ROS) in this process and the chemical identity of the ROS remain unknown. Methods and Results Transgenic mice with MLC-specific over-expression of catalase (TgCat-MLC mice) were created on a C57BL/6 background. Macrophage catalase activity was increased 3.4-fold compared to wild-type mice. After femoral artery ligation, LASER Doppler perfusion imaging revealed impaired perfusion recovery in TgCat-MLC mice. This was associated with fewer collateral vessels, as assessed by micro CT angiography, and decreased capillary density. Impaired functional recovery of the ischemic limb was also evidenced by a 50% reduction in spontaneous running activity. The deficient neovascularization was associated with a blunted inflammatory response, characterized by decreased macrophage infiltration of ischemic tissues, and lower mRNA levels of inflammatory markers such as tumor necrosis factor-α, osteopontin, and matrix mettaloproteinase-9. In vitro macrophage migration was impaired in TgCat-MLC mice, suggesting a role for H2O2 in regulating the ability of macrophages to infiltrate ischemic tissues. Conclusions MLC-derived H2O2 plays a key role in promoting neovascularization in response to ischemia and is a necessary factor for the development of ischemia-induced inflammation.