Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF.
Diabetes is a major risk factor for coronary and peripheral artery diseases. Although diabetic patients often present with advanced forms of these diseases, it is not known whether the compensatory mechanisms to vascular ischemia are affected in this condition. Accordingly, we sought to determine whether diabetes could: 1) impair the development of new collateral vessel formation in response to tissue ischemia and 2) inhibit cytokine-induced therapeutic neovascularization. Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 mice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler studies after the surgery. In NOD mice, measurement of the Doppler flow ratio between the ischemic and the normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery, Doppler flow ratio in the NOD mice was 0.49+/-0.04 versus 0.73+/-0.06 for the C57 mice (P< or =0.005). This impairment in blood flow recovery persisted throughout the duration of the study with Doppler flow ratio values at day 35 of 0.50+/-0.05 versus 0.90+/-0.07 in the NOD and C57 mice, respectively (P< or =0.001). CD31 immunostaining confirmed the laser Doppler data by showing a significant reduction in capillary density in the NOD mice at 35 days after surgery (302+/-4 capillaries/mm2 versus 782+/-78 in C57 mice (P< or =0.005). The reduction in neovascularization in the NOD mice was the result of a lower level of vascular endothelial growth factor (VEGF) in the ischemic tissues, as assessed by Northern blot, Western blot and immunohistochemistry. The central role of VEGF was confirmed by showing that normal levels of neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection of an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impairs endogenous neovascularization of ischemic tissues; 2) the impairment in new blood vessel formation results from reduced expression of VEGF; and 3) cytokine supplementation achieved by intramuscular adeno-VEGF gene transfer restores neovascularization in a mouse model of diabetes. (+info)
Granulocyte/macrophage colony-stimulating factor and interleukin-3 correct osteopetrosis in mice with osteopetrosis mutation.
Although young mice homozygous for the osteopetrosis (op) mutation usually developed prominent osteopetrosis, its severity was markedly reduced in aged op/op mice. This age-associated reversal of osteopetrosis was accompanied by the expansion of bone marrow cavities and increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive cells and of macrophages in the bone marrow. The TRAP-positive cells were mononuclear and developed ruffled borders and numerous vesicles, vacuoles, and granules. Enzyme-linked immunosorbent assay demonstrated a significant elevation of serum granulocyte/ macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 levels in the aged op/op mice. To examine whether GM-CSF and/or IL-3 could correct osteopetrosis in young op/op mice, 5 ng of recombinant murine (rm)GM-CSF and/or 100 ng of rmIL-3 were injected daily into young op/op mice. In these treated young op/op mice, the bone marrow cavities were expanded significantly at 2 weeks after administration, associated with significantly increased numbers of TRAP-positive cells and bone marrow macrophages. TRAP-positive cells increased in number with days after injection. These results suggest that GM-CSF and IL-3 induce the development of osteoclasts to correct osteopetrosis in the op/op mice with aging. (+info)
Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle.
The intermediate filament proteins nestin, vimentin, and desmin show a specific temporal expression pattern during the development of myofibers from myogenic precursor cells. Nestin and vimentin are actively expressed during early developmental stages to be later down-regulated, vimentin completely and nestin to minimal levels, whereas desmin expression begins later and is maintained in mature myofibers, in which desmin participates in maintaining structural integrity. In this study we have analyzed the expression levels and distribution pattern of nestin in intact and denervated muscle in rat and in human. Nestin immunoreactivity was specifically and focally localized in the sarcoplasm underneath neuromuscular junctions (NMJs) and in the vicinity of the myotendinous junctions (MTJs), ie, in regions associated with acetylcholine receptors (AChRs). This association prompted us to analyze nestin in neurogenically and myogenically denervated muscle. Immunoblot analysis disclosed a marked overall increase of accumulated nestin protein. Similar to the extrajunctional redistribution of AChRs in denervated myofibers, nestin immunoreactivity extended widely beyond the NMJ region. Re-innervation caused complete reversion of these changes. Our study demonstrates that the expression levels and distribution pattern of nestin are regulated by innervation, ie, signal transduction into myofibers. (+info)
The mechanism of the increasing action of TA-993, a new 1,5- benzothiazepine derivative, on limb blood flow in anesthetized dogs: selective suppression of sympathetic nerve activity.
TA-993, (-)-cis-3-acetoxy-5-(2-(dimethylamino)ethyl)-2, 3-di-hydro-8-methyl-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)one maleate, a new 1,5-benzothiazepine derivative with l-cis configuration, has a unique and selective increasing action on limb blood flow with little influence on arterial pressure besides an antiplatelet action. We studied the mechanism of increasing action of TA-993 on limb blood flow in anesthetized dogs. In a canine blood-perfused hindlimb preparation with a donor dog, TA-993 (100 microg/kg i.v.) did not increase femoral blood flow when administered to the donor dog, but did when administered to a recipient dog. TA-993 did not show the increasing action on femoral blood flow in the presence of hexamethonium or phentolamine, whereas it did in the presence of propranolol or atropine. TA-993 also showed a weak increasing effect on heart rate, which was inhibited by any one of these blockers. TA-993 (300 microg/kg i.v.) did not alter the phenylephrine (1-100 ng/kg i.a.)- or the talipexole (3-100 ng/kg i.a.)-induced increase in perfusion pressure in an autoperfused hindlimb. These results suggest that the increasing action of TA-993 on limb blood flow is mediated by the sympathetic nervous system but that the adrenergic receptors are not likely to be the central point of action of this new agent. There is a possibility that the mechanism of the increasing action on heart rate is different from that of its increasing action on limb blood flow. (+info)
The contribution of extraneuronal uptake to the trachea-blood vessel selectivity of beta-adrenoceptor stimulants in vitro in guinea-pigs.
1 The potencies relative to isoprenaline of isoetharine, tertiary butyl noradrenaline, salbutamol, orciprenaline, Me 506, rimiterol, fenoterol, carbuterol and terbutaline on isolated preparations of guinea-pig trachea and blood vessels (perfused hind limb) were determined. All the compounds were selective for trachea and selectivity values, i.e. relative potency on trachea divided by relative potency on hind limb, ranged from 2.3 to 21.4. 2 Responses to isoprenaline (the reference compound), tertiary butyl noradrenaline and isoetharine were potentiated on trachea by 50 muM phenoxybenzamine (PHB) and by other inhibitors of extraneuronal uptake (ENU). Under these conditions the selectivity values of all the compounds was close to unity. 3 Selectivity values were also close to unity if they were calculated from data obtained without ENU inhibition, provided that only those compounds not potentiated by PHB on trachea were used. 4 It is proposed that the trachea-blood vessel selectivity shown by beta-adrenoceptor stimulants can be caused by the influence of ENU upon them, rather than by their ability to distinguish between two beta2-adrenoceptors. 5 The suggestion that differences exist between beta2-adrenoceptors in respiratory and vascular smooth muscle is not supported by the in vitro experiments described. (+info)
Source of inappropriate receptive fields in cortical somatotopic maps from rats that sustained neonatal forelimb removal.
Previously this laboratory demonstrated that forelimb removal at birth in rats results in the invasion of the cuneate nucleus by sciatic nerve axons and the development of cuneothalamic cells with receptive fields that include both the forelimb-stump and the hindlimb. However, unit-cluster recordings from primary somatosensory cortex (SI) of these animals revealed few sites in the forelimb-stump representation where responses to hindlimb stimulation also could be recorded. Recently we reported that hindlimb inputs to the SI forelimb-stump representation are suppressed functionally in neonatally amputated rats and that GABAergic inhibition is involved in this process. The present study was undertaken to assess the role that intracortical projections from the SI hindlimb representation may play in the functional reorganization of the SI forelimb-stump field in these animals. The SI forelimb-stump representation was mapped during gamma-aminobutyric acid (GABA)-receptor blockade, both before and after electrolytic destruction of the SI hindlimb representation. Analysis of eight amputated rats showed that 75.8% of 264 stump recording sites possessed hindlimb receptive fields before destruction of the SI hindlimb. After the lesions, significantly fewer sites (13.2% of 197) were responsive to hindlimb stimulation (P < 0.0001). Electrolytic destruction of the SI lower-jaw representation in four additional control rats with neonatal forelimb amputation did not significantly reduce the percentage of hindlimb-responsive sites in the SI stump field during GABA-receptor blockade (P = 0.98). Similar results were obtained from three manipulated rats in which the SI hindlimb representation was silenced temporarily with a local cobalt chloride injection. Analysis of response latencies to sciatic nerve stimulation in the hindlimb and forelimb-stump representations suggested that the intracortical pathway(s) mediating the hindlimb responses in the forelimb-stump field may be polysynaptic. The mean latency to sciatic nerve stimulation at responsive sites in the GABA-receptor blocked SI stump representation of neonatally amputated rats was significantly longer than that for recording sites in the hindlimb representation [26.3 +/- 8.1 (SD) ms vs. 10.8 +/- 2.4 ms, respectively, P < 0.0001]. These results suggest that hindlimb input to the SI forelimb-stump representation detected in GABA-blocked cortices of neonatally forelimb amputated rats originates primarily from the SI hindlimb representation. (+info)
Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.
Pitx1 is a Bicoid-related homeodomain factor that exhibits preferential expression in the hindlimb, as well as expression in the developing anterior pituitary gland and first branchial arch. Here, we report that Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of the hindlimb, resulting in a limb that exhibits structural changes in tibia and fibula as well as patterning alterations in patella and proximal tarsus, to more closely resemble the corresponding forelimb structures. Deletion of the Pitx1 locus results in decreased distal expression of the hindlimb-specific marker, the T-box factor, Tbx4. On the basis of similar expression patterns in chick, targeted misexpression of chick Pitx1 in the developing wing bud causes the resulting limb to assume altered digit number and morphogenesis, with Tbx4 induction. We hypothesize that Pitx1 serves to critically modulate morphogenesis, growth, and potential patterning of a specific hindlimb region, serving as a component of the morphological and growth distinctions in forelimb and hindlimb identity. Pitx1 gene-deleted mice also exhibit reciprocal abnormalities of two ventral and one dorsal anterior pituitary cell types, presumably on the basis of its synergistic functions with other transcription factors, and defects in the derivatives of the first branchial arch, including cleft palate, suggesting a proliferative defect in these organs analogous to that observed in the hindlimb. (+info)
Trigeminal and carotid body inputs controlling vascular resistance in muscle during post-contraction hyperaemia in cats.
1. In anaesthetized cats, the effects of stimulation of the receptors in the nasal mucosa and carotid body chemoreceptors on vascular resistance in hindlimb skeletal muscle were studied to see whether the responses were the same in active as in resting muscle. The measurements of vascular resistance were taken, first, in resting muscle, and second, in the immediate post-contraction hyperaemic phase that followed a 30 s period of isometric contractions. 2. Stimulation of the receptors in the nasal mucosa caused reflex apnoea and vasoconstriction in muscle. The latter response was attenuated when the test was repeated during post-contraction hyperaemia. 3. Stimulations of the carotid bodies were made during a period of apnoea evoked reflexly by electrical stimulation of both superior laryngeal nerves. This apnoea prevented any effects of changes in respiration on the carotid body reflex vascular responses. Stimulation of the carotid bodies evoked hindlimb muscle vasoconstriction. In the post-contraction hyperaemic period, the response was reduced or abolished. A similar attenuation of the reflex vasoconstrictor responses occurred in decentralized muscles stimulated through their motor roots in the cauda equina. 4. Evidence is presented that the attenuation of the vasoconstrictor responses evoked by the two reflexes is a phenomenon localized to the contracting muscles themselves resulting from an interaction between sympathetic neuronal activity and the local production of metabolites. 5. The results are discussed in relation to the metabolic needs of tissues in relation to asphyxial defence mechanisms such as occur in the diving response. (+info)