Sonic hedgehog therapy in a mouse model of age-associated impairment of skeletal muscle regeneration.

Sonic hedgehog (Shh) is a morphogen regulating muscle development during embryogenesis. We have shown that the Shh pathway is postnatally recapitulated after injury and during regeneration of the adult skeletal muscle and regulates angiogenesis and myogenesis after muscle injury. Here, we demonstrate that in 18-month-old mice, there is a significant impairment of the upregulation of the Shh pathway that physiologically occurs in the young skeletal muscle after injury. Such impairment is even more pronounced in 24-month-old mice. In old animals, intramuscular therapy with a plasmid encoding the human Shh gene increases the regenerative capacities of the injured muscle, in terms of Myf5-positive cells, regenerating myofibers, and fibrosis. At the molecular level, Shh treatment increases the upregulation of the prototypical growth factors, insulin-like growth factor-1 and vascular endothelial growth factor. These data demonstrate that Shh increases regeneration after injury in the muscle of 24-month-old mice and suggest that the manipulation of the Shh pathway may be useful for the treatment of muscular diseases associated with aging.

Angiogenic impairment of the vascular endothelium: a novel mechanism and potential therapeutic target in muscular dystrophy.

OBJECTIVE: Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. APPROACH AND RESULTS: We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx-derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated ß-galactosidase activity. Mdx-derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell-derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. CONCLUSIONS: These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Combined therapy with sonic hedgehog gene transfer and bone marrow-derived endothelial progenitor cells enhances angiogenesis and myogenesis in the ischemic skeletal muscle.

We have previously demonstrated that sonic hedgehog (Shh) gene transfer improves angiogenesis in the setting of ischemia by upregulating the expression of multiple growth factors and enhancing the incorporation of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs is more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve regeneration after ischemia. We found a significantly higher capillary density in the Shh + EPC-treated muscles compared to the other experimental groups. We also found that Shh gene transfer increases the incorporation and survival of transplanted EPCs. Finally, we found a significantly higher number of regenerating myofibers in the ischemic muscles of mice receiving combined treatment with Shh and BM-derived EPCs. In summary, the combination of Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than each treatment individually and merits further investigation for its potential beneficial effects in ischemic diseases.

Pleiotropic beneficial effects of sonic hedgehog gene therapy in an experimental model of peripheral limb ischemia.

We have previously shown that the signaling pathway of the embryonic morphogen Sonic hedgehog (Shh) is recapitulated in the postnatal skeletal muscle in response to ischemia. We have also demonstrated that Shh is an indirect angiogenic agent upregulating various families of angiogenic growth factors and that Shh gene therapy improves angiogenesis and heart function in experimental models of myocardial ischemia. Based on these findings, we hypothesized that Shh gene therapy is beneficial in an experimental model of peripheral ischemia. We found that intramuscular (i.m.) treatment with a plasmid encoding the Shh human gene (phShh) increased blood flow, capillary density, and arteriole density in mice in which peripheral circulation of the hindlimb was disrupted by removal of the common femoral artery. Shh gene therapy also enhanced vasculogenesis, by increasing the number of circulating bone marrow (BM)-derived endothelial precursors and improving the contribution of these cells to the process of neovascularization. Finally, phShh treatment induced upregulation of prototypical angiogenic, arteriogenic, and vasculogenic factors, such as vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang-1), and stromal cell-derived factor-1 (SDF-1α). These data suggest that Shh gene therapy merits further investigation for its ability to trigger the expression of potent trophic factors and stimulate pleiotropic aspects of neovascularization in the setting of ischemia.

Combined effect of inflammatory gene polymorphisms and the risk of ischemic stroke in a prospective cohort of subjects with type 2 diabetes: a Go-DARTS study.

OBJECTIVE: We have previously observed that genetic profiles determined by the combination of five functionally significant single nucleotide polymorphisms (SNPs) (rs1800795, rs5498, rs5361, rs1024611, and rs679620) of genes encoding prototypical inflammatory molecules are associated with history of ischemic stroke. Here we tested the ability of this multigenic model to predict stroke risk in a large population-based prospective cohort of subjects with type 2 diabetes. RESEARCH DESIGN AND METHODS: This study was conducted using a prospective cohort of individuals with type 2 diabetes participating in the Go-DARTS (Genetics of Diabetes Audit and Research in Tayside Scotland) study, which includes genetic and clinical information of patients with diabetes within the Tayside region of Scotland, U.K. The above-mentioned inflammatory SNPs were investigated in 2,182 Go-DARTS participants. We created an inflammatory risk score (IRS), ranging from 0 to 5, according to the number of "at-risk" genotypes concomitantly carried by a given individual. The primary outcome was the occurrence of fatal or nonfatal stroke of any kind. Mean follow-up time was 6.2 ± 1.1 years. RESULTS: The incidence of stroke increased according to the IRS. The IRS was significantly and independently associated with increased stroke risk after adjustment for other conventional risk factors (hazard ratio 1.34 [95% CI 1.1-1.7]; P = 0.009). The highest hazard ratio for stroke was found in subjects concomitantly carrying > 3 proinflammatory variations and in subjects without previous cardiovascular diseases. CONCLUSIONS: This large prospective cohort study provides evidence that SNPs of genes encoding prototypical inflammatory molecules may be used to create multigenic models that predict stroke risk in subjects with type 2 diabetes.

Peroxisome proliferator-activated receptor alpha is crucial for iloprost-induced in vivo angiogenesis and vascular endothelial growth factor upregulation.

We have previously demonstrated that iloprost, a stable prostacyclin (PGI(2)) analogue, induces angiogenesis in vivo, through a vascular endothelial growth factor (VEGF)-dependent mechanism. In this study, we demonstrate that iloprost-induced angiogenesis and VEGF upregulation are modulated by peroxisome proliferator-activated receptor-alpha (PPARalpha), a ligand-inducible transcription factor that belongs to the nuclear hormone receptor superfamily and plays multiple biological activities in the vascular system. We show that iloprost is unable to induce angiogenesis in mice lacking the PPARalpha gene (PPARalpha-/- mice). Likewise, iloprost-induced VEGF upregulation is absent in PPARalpha-/- mice. In contrast, iloprost induces a robust angiogenic response in wild-type mice, along with local upregulation of VEGF. Importantly, mice lacking the PPARalpha gene exhibit a normal angiogenic response to VEGF, indicating that the absence of PPARalpha does not result in a general impairment of angiogenesis, but specifically affects the ability of iloprost to induce angiogenesis. Our data demonstrate unexpected functional relationships between the PGI(2) system and the PPAR signaling pathway and shed new light on the molecular mechanisms involved in iloprost-induced angiogenesis.

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.

Selective activation of peroxisome proliferator-activated receptor (PPAR)alpha and PPAR gamma 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 PPAR alpha and PPAR gamma stimulates neoangiogenesis. RESEARCH DESIGN AND METHODS: We used selective synthetic PPAR alpha and PPAR gamma agonists and investigated their angiogenic potentials in vitro and in vivo. RESULTS: Activation of PPAR alpha and PPAR gamma 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 PPAR alpha and PPAR gamma agonists are used in the murine corneal angiogenic model. No vascular growth is detectable when PPAR alpha and PPAR gamma agonists are respectively used in PPAR alpha knockout mice and mice treated with a specific PPAR gamma inhibitor, demonstrating that this angiogenic response is PPAR mediated. PPAR alpha- and PPAR gamma-induced angiogenesis is associated with local VEGF production and does not differ in extent and morphology from that induced by VEGF. In addition, PPAR alpha- and PPAR gamma-induced in vitro and in vivo angiogenesis may be significantly decreased by inhibiting VEGF activity. Finally, in corneas treated with PPAR alpha and PPAR gamma agonists, there is increased phosphorylation of endothelial nitric oxide synthase and Akt. CONCLUSIONS: These findings demonstrate that PPAR alpha and PPAR gamma activation stimulates neoangiogenesis through a VEGF-dependent mechanism. Neoangiogenesis is a crucial pathological event in type 2 diabetes. The ability of PPAR alpha and PPAR gamma agonists to induce neoangiogenesis might have important implications for the clinical and therapeutic management of type 2 diabetes.

Intraneuronal beta-amyloid expression downregulates the Akt survival pathway and blunts the stress response.

Early events in Alzheimer's disease (AD) pathogenesis implicate the accumulation of beta-amyloid (Abeta) peptide inside neurons in vulnerable brain regions. However, little is known about the consequences of intraneuronal Abeta on signaling mechanisms. Here, we demonstrate, using an inducible viral vector system to drive intracellular expression of Abeta42 peptide in primary neuronal cultures, that this accumulation results in the inhibition of the Akt survival signaling pathway. Induction of intraneuronal Abeta42 expression leads to a sequential decrease in levels of phospho-Akt, increase in activation of glycogen synthase kinase-3beta, and apoptosis. Downregulation of Akt also paralleled intracellular Abeta accumulation in vivo in the Tg2576 AD mouse model. Overexpression of constitutively active Akt reversed the toxic effects of Abeta through a mechanism involving the induction of heat shock proteins (Hsps). We used a small-interfering RNA approach to explore the possibility of a link between Akt activity and Hsp70 expression and concluded that neuroprotection by Akt could be mediated through downstream induction of Hsp70 expression. These results suggest that the early dysfunction associated with intraneuronal Abeta accumulation in AD involve the associated impairments of Akt signaling and suppression of the stress response.

MRI-guided ultrasonic heating allows spatial control of exogenous luciferase in canine prostate.

The need for efficient and controlled delivery is one of the major obstacles to clinical use of gene therapy. In this study, we investigated the use of magnetic resonance imaging-monitored ultrasound (US) to induce expression of luciferase after local injection of the construct Ad-HSP-Luc, an adenoviral vector containing a transgene encoding firefly luciferase under the control of the human hsp70B promoter. The hsp promoter allows induction of the associated transgene only in areas that are subsequently heated after infection. US imaging was used to guide the injection of purified virus into both lobes of the prostates of three beagles. At 48 h after injection, the left lobe of the prostate was heated using a 1.5-MHz US transducer driven by a multichannel radiofrequency system and employing an magnetic resonance imaging guidance system. High levels of luciferase expression were observed only in areas exposed to ultrasonic heating. This study demonstrates the feasibility of using ultrasonic heating to control transgene expression spatially using a minimally-invasive approach.

Age-dependent VEGF expression and intraneural neovascularization during regeneration of peripheral nerves.

The physiologic ability of peripheral nerves to regenerate after injury is impaired with aging. However, the mechanisms responsible for this phenomenon are still incompletely characterized. In this study, we investigated whether aging influences the intraneural angiogenic response that occurs after injury and during regeneration of peripheral nerves. We performed a crush injury of the sciatic nerve in old and senescence accelerated mice and found that the peripheral nerves of these animals are unable to locally upregulate vascular endothelial growth factor (VEGF), a prototypical angiogenic cytokine, after injury and have substantial deficits in mounting an appropriate intraneural angiogenic response during nerve regeneration. Our findings provide new evidence of possible interdependent relationships between aging, VEGF, angiogenesis, and nerve regeneration and suggest that vascular abnormalities might play a role in aging-associated neurological dysfunction, with potentially important fundamental and clinical implications.

Heat shock protein 70 participates in the neuroprotective response to intracellularly expressed beta-amyloid in neurons.

Intracellular beta-amyloid 42 (Abeta42) accumulation is increasingly recognized as an early event in the pathogenesis of Alzheimer's disease (AD). We have developed a doxycycline-inducible adenoviral-based system that directs intracellular Abeta42 expression and accumulation into the endoplasmic reticulum of primary neuronal cultures in a regulated manner. Abeta42 exhibited a perinuclear distribution in cell bodies and an association with vesicular compartments. Virally expressed intracellular Abeta42 was toxic to neuronal cultures 24 hr after induction in a dose-dependent manner. Abeta42 expression prompted the rapid induction of stress-inducible Hsp70 protein in neurons, and virally mediated Hsp70 overexpression rescued neurons from the toxic effects of intracellular Abeta accumulation. Together, these results implicate the cellular stress response as a possible modulator of Abeta-induced toxicity in neuronal cultures.

Peripheral nerve ischemia: apolipoprotein E deficiency results in impaired functional recovery and reduction of associated intraneural angiogenic response.

Apolipoprotein E-knockout (apoE KO) mice have peripheral sensory nerve defects, reduced and delayed response to noxious thermal stimuli, abnormal morphology of unmyelinated fibers, and impaired blood-nerve and blood-brain barriers. In this study, we show that, compared to wild-type mice, peripheral nerves of apoE KO mice have impaired ability to respond to ischemia, as demonstrated by measurement of motor and sensory conduction velocity. In addition, mice lacking apoE exhibit a deficit of reinnervation of ischemic epidermis, evaluated by immunofluorescent staining for the pan-neuronal marker PGP 9.5. Also regional nerve blood flow, measured by laser Doppler, and intraneural angiogenesis after ischemia are significantly compromised in apoE-deficient mice. Finally, upregulation of the angiogenic cytokine vascular endothelial growth factor (VEGF), which physiologically occurs after ischemia in the peripheral nerve of wild-type mice, is severely impaired in apoE KO mice. Among the several neural defects that have already been described in mice lacking apoE, this is the first demonstration that functional recovery to ischemia is impaired in the peripheral nerves of these animals. This deficit is mirrored by the inability of upregulating VEGF and mounting an appropriate intraneural angiogenic response following injury. These findings provide new evidence of possible interdependent relationships between VEGF, angiogenesis, and nerve function and regeneration and may provide new important information on the role of apoE in the nervous system.

IL-6 and matrix metalloproteinase-1 are regulated by the cyclin-dependent kinase inhibitor p21 in synovial fibroblasts.

During the pathogenesis of rheumatoid arthritis (RA), the synovial fibroblasts increase in number and produce proinflammatory cytokines and matrix metalloproteinases (MMPs) that function to promote inflammation and joint destruction. Recent investigations have suggested that cell cycle activity and inflammation may be linked. However, little is known about the mechanisms responsible for the coordinate regulation of proliferation and the expression of proinflammatory molecules in RA synovial fibroblasts. Here, we demonstrate a 50 +/- 10% decrease in the expression of p21, a cell cycle inhibitor, in the synovial fibroblast population from RA compared with osteoarthritis (OA) synovial tissue. Moreover, p21 positivity in the synovial fibroblasts inversely correlates with medium synovial lining thickness (r = -0.76; p < 0.02). The expression of p21 is also reduced in isolated RA synovial fibroblasts compared with OA synovial fibroblasts. Adenovirus-mediated delivery of p21 (Ad-p21) arrests both RA and OA synovial fibroblasts in the G(0)/G(1) phase of the cell cycle without inducing cytotoxicity. However, the spontaneous production of IL-6 and MMP-1 is suppressed only in the Ad-p21-infected RA synovial fibroblasts, indicating a novel role for p21 in RA. Analyses of p21-deficient mouse synovial fibroblasts reveal a 100-fold increase in IL-6 protein and enhance IL-6 and MMP-3 mRNA. Restoration of p21, but not overexpression of Rb, which also induces G(0)/G(1) cell cycle arrest, decreases IL-6 synthesis in p21-null synovial fibroblasts. Furthermore, in RA synovial fibroblasts the ectopic expression of p21 reduces activation of the AP-1 transcription factor. Additionally, p21-null synovial fibroblasts display enhanced activation of AP-1 compared with wild-type synovial fibroblasts. These data suggest that alterations in p21 expression may activate AP-1 leading to enhanced proinflammatory cytokine and MMP production and development of autoimmune disease.

Spatial and temporal control of transgene expression through ultrasound-mediated induction of the heat shock protein 70B promoter in vivo.

Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) or Fas ligand (Adeno-HSP-FasL) under the control of the heat shock protein 70B (hsp70B) promoter. Cultures infected with Adeno-HSP-Luc transiently expressed high levels of luciferase after heat shock. When cultures infected with Adeno-HSP-FasL were maintained at 37 degrees C, no transgene expression was observed, but when cultures were exposed to heat stress, transgene expression resulted in apoptotic cell death. In vivo, transgene expression was induced by ultrasound-mediated heating of adenovirus-infected tissue. In mice or rats injected with the Adeno-HSP-Luc construct, high levels of localized expression of luciferase activity were observed in regions subjected to ultrasound-mediated irradiation. Adeno-HSP-FasL was administered systemically to mice via the tail vein to evaluate safety. Animals receiving Adeno-HSP-FasL in the absence of ultrasound treatment did not display liver toxicity, whereas animals receiving ultrasound treatment to induce the expression of Fas ligand from the hsp70B promoter had significant increases in serum levels of liver enzymes. These data demonstrate that combining the inducible hsp70B promoter with ultrasound induction allows safe local expression of cytotoxic genes with possible therapeutic utility.