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 1 receptor gene ( P 1 receptors blunted ischemia-induced angiogenesis ( P 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 P P 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

Multiple effects of high mobility group box protein 1 in skeletal muscle regeneration

OBJECTIVE - High mobility group box 1 protein (HMGB1) is a cytokine released by necrotic and inflammatory cells in response to injury. We examined the role of HMGB1 in skeletal muscle regeneration after hindlimb ischemia. METHODS AND RESULTS - Unilateral hindlimb ischemia was induced in mice by femoral artery dissection. HMGB1 levels increased in regenerating skeletal muscle and the blockade of endogenous HMGB1 by the administration of its truncated form, the BoxA, resulted in the reduction of vessel density. In contrast, intramuscular administration of HMGB1 enhanced perfusion and increased the number of regenerating fibers. To separately study the myogenic and the angiogenic effects of HMGB1, in vitro experiments were performed with isolated myoblasts and endothelial cells. Myoblasts were found to express the HMGB1 receptor RAGE and TLR4 which were downregulated during in vitro myogenic differentiation. HMGB1 was extracellularly released by differentiated myoblasts and exerted a chemotactic activity on myogenic cells. This effect was partially dependent on RAGE and was inhibited by BoxA treatment. Finally, HMGB1 stimulated tubular-like structure formation by endothelial cells through the activation of extracellular signal-regulated kinase (ERK) and JNK signal transduction pathways. CONCLUSIONS - HMGB1 plays a role in skeletal muscle regeneration modulating, in an autocrine-paracrine manner, myoblast and endothelial cell functions

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

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 &lt;0.01). In B 2 +/− mice, BP elevation was delayed. At 40 days, the heart rate was higher ( P &lt;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 &lt;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. </jats:p

SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit + stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting α 2, α 4, and α 5 integrin-mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit + cells, while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16 INK4 restored such differentiation, suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection, plasma SDF-1 levels were up-regulated, while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage age of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RMA and protein level was also observed. Finally, using an in vivo assay such as injection of matrigel plugs, we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit + cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells. © 2004 by The American Society of Hematology

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