BAG3 (Bcl-2-Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy.

BACKGROUND: Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1, that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. METHODS: We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. RESULTS: We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6-Lsq1-3). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein- (n=29) expressing animals. BAG3Ile81, but not BAG3Met81, improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus-BAG3Ile81 (n=9), but not BAG3Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3Met81, BAG3Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. CONCLUSIONS: Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway.

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1's role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.

Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment.

Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.

Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature.

Retinal and choroidal neovascularization (NV) and vascular leakage contribute to visual impairment in several common ocular diseases. The angiopoietin/TIE2 (ANG/TIE2) pathway maintains vascular integrity, and negative regulators of this pathway are potential therapeutic targets for these diseases. Here, we demonstrated that vascular endothelial-protein tyrosine phosphatase (VE-PTP), which negatively regulates TIE2 activation, is upregulated in hypoxic vascular endothelial cells, particularly in retinal NV. Intraocular injection of an anti-VE-PTP antibody previously shown to activate TIE2 suppressed ocular NV. Furthermore, a small-molecule inhibitor of VE-PTP catalytic activity (AKB-9778) activated TIE2, enhanced ANG1-induced TIE2 activation, and stimulated phosphorylation of signaling molecules in the TIE2 pathway, including AKT, eNOS, and ERK. In mouse models of neovascular age-related macular degeneration, AKB-9778 induced phosphorylation of TIE2 and strongly suppressed NV. Ischemia-induced retinal NV, which is relevant to diabetic retinopathy, was accentuated by the induction of ANG2 but inhibited by AKB-9778, even in the presence of high levels of ANG2. AKB-9778 also blocked VEGF-induced leakage from dermal and retinal vessels and prevented exudative retinal detachments in double-transgenic mice with high expression of VEGF in photoreceptors. These data support targeting VE-PTP to stabilize retinal and choroidal blood vessels and suggest that this strategy has potential for patients with a wide variety of retinal and choroidal vascular diseases.

Ca2+-dependent, stimulus-specific modulation of the plasma membrane Ca2+ pump in hippocampal neurons.

The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in restoring Ca(2+) to basal levels following transient elevation by neuronal activity. Here we examined the effects of various stimuli that increase [Ca(2+)](i) on PMCA-mediated Ca(2+) clearance from hippocampal neurons. We used indo-1-based microfluorimetry in the presence of cyclopiazonic acid to study the rate of PMCA-mediated recovery of Ca(2+) elevated by a brief train of action potentials. [Ca(2+)](i) recovery was described by an exponential decay and the time constant provided an index of PMCA-mediated Ca(2+) clearance. PMCA function was assessed before and for >or=60 min following a 10-min priming stimulus of either 100 microM N-methyl-d-aspartate (NMDA), 0.1 mM Mg(2+) (reduced extracellular Mg(2+) induces intense excitatory synaptic activity), 30 mM K(+), or control buffer. Recovery kinetics slowed progressively following priming with NMDA or 0.1 mM Mg(2+); in contrast, Ca(2+) clearance initially accelerated and then slowly returned to initial rates following priming with 30 mM K(+)-induced depolarization. Treatment with 10 muM calpeptin, an inhibitor of the Ca(2+) activated protease calpain, prevented the slowing of kinetics observed following treatment with NMDA but had no affect on the recovery kinetics of control cells. Calpeptin also blocked the rapid acceleration of Ca(2+) clearance following depolarization. In calpeptin-treated cells, 0.1 mM Mg(2+) induced a graded acceleration of Ca(2+) clearance. Thus in spite of producing comparable increases in [Ca(2+)](i), activation of NMDA receptors, depolarization-induced activation of voltage-gated Ca(2+) channels and excitatory synaptic activity each uniquely affected Ca(2+) clearance kinetics mediated by the PMCA.

gamma-Secretase activity modulates store-operated Ca2+ entry into rat sensory neurons.

Presenilin-1 is required for gamma-secretase activity, which participates in Notch receptor processing, the pathogenesis of Alzheimer's disease and the modulation of Ca(2+) signaling. We tested the hypothesis that gamma-secretase proteolytic activity modulates store-operated Ca(2+) entry (SOCE) in rat dorsal root ganglion (DRG) neurons. Depletion of intracellular Ca(2+) stores by blocking the endoplasmic reticulum (ER) Ca(2+) pump with cyclopiazonic acid (CPA) evoked a transient increase in [Ca(2+)](i) but no sustained Ca(2+) influx. However, in cells expressing a dominant negative presenilin-1 mutant (PS1-D257A), gamma-secretase activity was inhibited and treatment with CPA evoked sustained Ca(2+) influx. Similarly, pharmacologic inhibition of gamma-secretase with DAPT for 48h enhanced SOCE. SKF96365, an inhibitor of store-operated channels, blocked SOCE in cells expressing PS1-D257A. Thus, gamma-secretase proteolytic activity regulates a SOCE pathway in sensory neurons.