Sonic hedgehog gene therapy increases the ability of the dystrophic skeletal muscle to regenerate after injury.

The Hedgehog (Hh) pathway is a crucial regulator of muscle development during embryogenesis. We have previously demonstrated that Sonic hedgehog (Shh) regulates postnatal myogenesis in the adult skeletal muscle both directly, by acting on muscle satellite cells, and indirectly, by promoting the production of growth factors from interstitial fibroblasts. Here, we show that in mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, progression of the dystrophic pathology corresponds to progressive inhibition of the Hh signaling pathway in the skeletal muscle. We also show that the upregulation of the Hh pathway in response to injury and during regeneration is significantly impaired in mdx muscle. Shh treatment increases the proliferative potential of satellite cells isolated from the muscles of mdx mice. This treatment also increases the production of proregenerative factors, such as insulin-like growth factor-1 and vascular endothelial growth factor, from fibroblasts isolated from the muscle of mdx mice. In vivo, overexpression of the Hh pathway using a plasmid encoding the human Shh gene promotes successful regeneration after injury in terms of increased number of proliferating myogenic cells and newly formed myofibers, as well as enhanced vascularization and decreased fibrosis.

Proteomic profile of differentially expressed plasma proteins from dystrophic mice and following suberoylanilide hydroxamic acid treatment.

PURPOSE: Histone Deacetylase Inhibitors (DI) ameliorates dystrophic muscle regeneration restoring muscular strength in the mdx mouse model of Duchenne muscular dystrophy (DMD). The further development of these compounds as drugs for DMD treatment is currently hampered by the lack of knowledge about DIs effect in large dystrophic animal models and that of suitable biomarkers to monitor their efficacy. EXPERIMENTAL DESIGN: In this study we applied proteomic analysis to identify differentially expressed proteins present in plasma samples from mdx mice treated with the Suberoylanilide hydroxamic acid (SAHA) and relative normal controls (WT). RESULTS: Several differentially expressed proteins were identified between untreated wild type and mdx mice. Among these, fibrinogen, epidermal growth factor 2 receptor, major urinary protein and glutathione peroxidase 3 (GPX3) were constitutively up-regulated in mdx, while complement C3, complement C6, gelsolin, leukaemia inhibitory factor receptor (LIFr), and alpha 2 macroglobulin were down-regulated compared to WT mice. SAHA determined the normalization of LIFr and GPX3 protein level while apoliprotein E was de novo up-regulated in comparison to vehicle-treated mdx mice. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, these data unravel potential serological disease biomarkers of mdx that could be useful to monitor muscular dystrophy response to DI treatment.

Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors.

Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles. We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and alpha-sarcoglycan (alpha-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies.

Renal phenotype of low kallikrein rats.

BACKGROUND: Renal kallikrein has been linked with inheritance of arterial hypertension and with sensitivity to drug nephrotoxicity. Identification of a cause--effect relationship between low kallikrein and intermediate phenotypes has been hampered by the lack of adequate animal models. METHODS: Kallikrein was measured in tissues obtained from rats inbred for low urinary kallikrein excretion (LKR) and wild-type controls. Blood pressure and indices of myocardial contractility were recorded via an intraventricular cannula connected to a transducer. The functional relevance of endogenous angiotensin II (Ang II) in LKR was explored by determining the effect of Ang II subtype 1 (AT(1)) receptor blockade on glomerular filtration rate, renal blood flow, and urinary sodium excretion. In addition, sensitivity to gentamycin-induced nephrotoxicity was evaluated. RESULTS: Kallikrein activity was reduced by 60% in the kidney of LKR (P < 0.01), whereas it was increased in the heart (P < 0.05) and was unaltered in the pancreas, liver, and salivary glands. Heart rate and myocardial contractility were reduced, and the mean blood pressure (MBP) was increased in LKR as compared with controls (P < 0.05). LKR exhibited polydipsia, polyuria, glomerular hyperfiltration, and reduced fractional sodium excretion under basal conditions and impaired renal vasodilation in response to volume expansion. These functional alterations were significantly attenuated by AT(1) receptor blockade. Gentamycin reduced the glomerular filtration rate in LKR, but not in controls. CONCLUSIONS: In LKR, unopposed activity of Ang II appears to be responsible for increased glomerular hydrostatic pressure and augmented tubular reabsorption. Balance between the kallikrein-kinin and renin-angiotensin systems is essential for normal renal function.

Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND: Human tissue kallikrein (HK) releases kinins from kininogen. We investigated whether adenovirus-mediated HK gene delivery is angiogenic in the context of ischemia. METHODS AND RESULTS: Hindlimb ischemia, caused by femoral artery excision, increased muscular capillary density (P:<0.001) and induced the expression of kinin B(1) receptor gene (P:<0.05). Pharmacological blockade of B(1) receptors blunted ischemia-induced angiogenesis (P:<0.01), whereas kinin B(2) receptor antagonism was ineffective. Intramuscular delivery of adenovirus containing the HK gene (Ad. CMV-cHK) enhanced the increase in capillary density caused by ischemia (969+/-32 versus 541+/-18 capillaries/mm(2) for control, P:<0.001), accelerated blood flow recovery (P:<0.01), and preserved energetic charge of ischemic muscle (P:<0.01). Chronic blockade of kinin B(1) or B(2) receptors prevented HK-induced angiogenesis. CONCLUSIONS: HK gene delivery enhances the native angiogenic response to ischemia. Angiogenesis gene therapy with HK might be applicable to peripheral occlusive vascular disease.

Adenovirus-mediated human tissue kallikrein gene delivery induces angiogenesis in normoperfused skeletal muscle.

We investigated whether local delivery of the tissue kallikrein gene induces angiogenesis in normoperfused mouse hindlimb muscles. Intramuscular injection of adenovirus containing the human tissue kallikrein gene under the control of a cytomegalovirus enhancer/promoter sequence resulted in local production and release of recombinant human tissue kallikrein, whereas transgene expression was absent in muscles of the contralateral hindlimb. Angiogenesis in infected muscles was documented by histological evidence of increased capillary density. In contrast, no angiogenic effect was seen either in the ipsilateral gastrocnemius or contralateral hindlimb muscles. Neovascularization was associated with a transient increase in muscular blood flow as determined by laser Doppler flowmetry. We also investigated the mechanisms of kallikrein-induced angiogenesis. We found that the angiogenic response to kallikrein was abolished by chronic blockade of the kinin B(1) or B(2) receptor or by inhibition of nitric oxide synthase. In addition, inhibition of cyclooxygenase-2 by nimesulide significantly reduced kallikrein-induced effects. These results indicate that (1) human tissue kallikrein acts as an angiogenic factor in normoperfused skeletal muscle and (2) nitric oxide and prostacyclin are essential mediators of kallikrein-induced angiogenesis. Our findings provide new insights into the role of the tissue kallikrein-kinin system in vascular biology.

Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ischemia.

BACKGROUND: Administration of angiogenic factors stimulates neovascularization in ischemic tissues. However, there is no evidence that angiogenesis can be induced in normoperfused skeletal muscles. We tested the hypothesis that adenovirus-mediated intramuscular (IM) gene transfer of the 121-amino-acid form of vascular endothelial growth factor (AdCMV.VEGF(121)) could stimulate neovascularization in nonischemic skeletal muscle and consequently attenuate the hemodynamic deficit secondary to surgically induced ischemia. METHODS AND RESULTS: Rabbits and rats received IM injections of AdCMV.VEGF(121), AdCMV.Null, or saline in the thigh, 4 weeks (rabbits) or 2 weeks (rats) before femoral artery removal in the injected limb. In unoperated rats, at the site of injection of AdCMV.VEGF(121), we found 96% and 29% increases in length density of arterioles and capillaries, respectively. Increased tissue perfusion (TP) to the ischemic limb in the AdCMV.VEGF(121) group was documented, as early as day 1 after surgery, by improved blood flow to the ischemic gastrocnemius muscle measured by radioactive microspheres (AdCMV.VEGF(121)=5.69+/-0.40, AdCMV.Null=2.97+/-0.50, and saline=2.78+/-0.43 mL x min(-1) x 100 g(-1), P<0.001), more angiographically recognizable collateral vessels (angioscore) (AdCMV. VEGF(121)=50.58+/-1.48, AdCMV.Null=29.08+/-4.22, saline=11.83+/-1.90, P<0.0001), and improvement of the bioenergetic reserve of the gastrocnemius muscle as assessed by (31)P NMR spectroscopy. Follow-up studies showed that superior TP to the ischemic limb in the AdCMV.VEGF(121) group persisted until it was equalized by spontaneous collateral vessel development in untreated animals. CONCLUSIONS: IM administration of AdCMV.VEGF(121) stimulates angiogenesis in normoperfused skeletal muscles, and the newly formed vessels preserve TP after induction of ischemia.

Adenovirus-mediated human tissue kallikrein gene delivery inhibits neointima formation induced by interruption of blood flow in mice.

Tissue kallikrein cleaves kininogen to produce vasoactive kinin peptides. Binding of kinins to bradykinin B(2) receptors on vascular endothelial cells stimulates the release of nitric oxide and prostacyclin, thus activating the cGMP and cAMP pathways. In this study, we evaluated the effects of adenovirus-mediated human tissue kallikrein gene (Ad.CMV-cHK) delivery in a mouse model of arterial remodeling induced by permanent alteration in shear stress conditions. Mice underwent ligature of the left common carotid artery and were injected intravenously with saline or 1.8 x 10(9) plaque-forming units of Ad.CMV-cHK or control virus (Ad.CMV-LacZ). Fourteen days after surgery, morphometric analysis revealed that Ad. CMV-cHK reduced neointima formation by 52% (P<0.05) compared with Ad. CMV-LacZ. Expression of human tissue kallikrein (HK) mRNA was detected in mouse carotid artery, aorta, kidney, heart, and liver, and recombinant HK was present in the urine and plasma of mice receiving HK gene. Kallikrein gene transfer resulted in increases in urinary kinin, cGMP, and cAMP levels. The protective action of Ad. CMV-cHK on neointima formation was significantly reduced (P<0.05) in mice with knockout of the kinin B(2) receptor gene compared with wild-type control mice (J129Sv mice). In contrast, the effect of Ad. CMV-cHK was amplified (P<0.05) in transgenic mice overexpressing human B(2) receptor compared with wild-type control mice (c57/Bl6 mice). Thus, the inhibitory effect of recombinant kallikrein on structural alterations caused by the interruption of blood flow appears to be mediated by the B(2) receptor. These results provide new insight into the role of the tissue kallikrein-kinin system in vascular remodeling and suggest the application of HK gene therapy to treat restenosis and atherosclerosis.

Dilated and failing cardiomyopathy in bradykinin B(2) receptor knockout mice.

BACKGROUND: The activation of B(2) receptors by kinins could exert cardioprotective effects in myocardial ischemia and heart failure. METHODS AND RESULTS: To test whether the absence of bradykinin B(2) receptors may affect cardiac structure and function, we examined the developmental changes in blood pressure (BP), heart rate, and heart morphology of bradykinin B(2) receptor gene knockout (B(2)(-/-)), heterozygous (B(2)(+/-)), and wild-type (B(2)(+/+)) mice. The BP of B(2)(-/-) mice, which was still normal at 50 days of age, gradually increased, reaching a plateau at 6 months (136+/-3 versus 109+/-1 mm Hg in B(2)(+/+), P<0.01). In B(2)(+/-) mice, BP elevation was delayed. At 40 days, the heart rate was higher (P<0.01) in B(2)(-/-) and B(2)(+/-) than in B(2)(+/+) mice, whereas the left ventricular (LV) weight and chamber volume were similar among groups. Thereafter, the LV growth rate of B(2)(-/-) and B(2)(+/-) mice was accelerated, leading at 360 days to a LV weight-to-body weight ratio that was 9% and 17% higher, respectively, than that of B(2)(+/+) mice. In B(2)(-/-) mice, hypertrophy was associated with a marked chamber dilatation (42% larger than that of B(2)(+/+) mice), an elevation in LV end-diastolic pressure (25+/-3 versus 5+/-1 mm Hg in B(2)(+/+) mice, P<0.01), and reparative fibrosis. CONCLUSIONS: The disruption of the bradykinin B(2) receptor leads to hypertension, LV remodeling, and functional impairment, implying that kinins are essential for the functional and structural preservation of the heart.