Lack of effect of bone morphogenetic protein 2 and 4 gene polymorphisms on bone density in postmenopausal Turkish women.

We investigated the effect of bone morphogenetic protein 2 and 4 (BMP-2 and -4) gene polymorphisms on bone density in postmenopausal Turkish women with osteoporosis. The frequency of single-nucleotide polymorphisms (SNPs) of BMP-2 and -4 genes was analyzed in 101 osteoporotic-postmenopausal women and 52 postmenopausal women with positive bone mineral density scores. We evaluated the frequency of the thymine→cytosine nucleotide variation at position 538 for BMP-4 and the transposition of adenine→thymine at codon 190 for BMP-2, with PCR. The proportions of genotypes observed for the BMP-2 SNP in the osteoporotic group were AA (47.5%), AT (39.6%), TT (12.9%), and in the non-osteoporotic group they were AA (48.1%), AT (40.4%), TT (11.5%). The corresponding frequencies for the BMP-4 SNP in the osteoporotic group were TT (30.7%), TC (45.5%), CC (23.8%), and in the non-osteoporotic group they were TT (26.9%), TC (40.4%), CC (32.7%). There were no significant differences in the frequencies of these genotypes between the patient and control groups. We conclude that genetic variations in BMP-2 and -4 do not substantially contribute to lumbar spine bone mineral density in postmenopausal Turkish women.

Analysis of vascular endothelial growth factor gene 936 C/T polymorphism in patients with familial Mediterranean fever.

Vascular endothelial growth factor (VEGF) is a cytokine that promotes endothelial cell proliferation, leucocyte chemotaxis and expression of adhesion molecules and is a major mediator of vascular permeability. It has been demonstrated that VEGF directly activates neutrophils and it could promote acute recruitment of leucocytes. It is known that neutrophils are the major cell population involved in acute inflammation in familial Mediterranean fever (FMF) and the role of VEGF in these cells may be crucial. The aim of this study was to investigate whether the 936 C/T functional polymorphism of the VEGF gene is associated with susceptibility to FMF and its relationship with the main clinical features of the disease. Polymerase chain reaction-restriction fragment length polymorphism technique was used to determine 936 C/T polymorphism within the VEGF gene in 75 patients with FMF and 122 non-related healthy controls. Genotype and allele frequencies of the VEGF 936 C/T polymorphism between patients with FMF and healthy control groups were not significantly different (OR = 0.74, 95% CI = 0.40-1.37, P = 0.335 for CT genotype; OR = 1.11, 95% CI = 0.67-1.83, P = 0.700, for T allele). Although VEGF 936 TT genotype was found to be more frequent in patients with FMF than in healthy controls (6.7% vs. 1.6%, respectively), the difference was not significant (OR = 4.28, 95% CI = 0.81-22.67, P = 0.108). No associations were found between the studied polymorphism and either the clinical features such as arthritis, abdominal pain, pleuritis, myalgia, arthralgia and erysipelas-like erythema of the disease or the four common studied exon 10 mutations (M694V, M680I, V726A, M694I) of the Mediterranean fever gene. Present results suggest that VEGF gene 936 C/T polymorphism does not seem to be associated with susceptibility to FMF and its clinical manifestations.

Cold exposure differentially stimulates angiogenesis in glycolytic and oxidative muscles of rats and hamsters.

Control (normothermic) and cold-acclimated (environmental temperature gradually reduced from 20 to 5 degrees C for 4 weeks) groups of male rats and hamsters were compared to elucidate the nature of angiogenesis in oxidative and glycolytic muscles of these species during progressive cold exposure. Skeletal muscle capillarity and fibre cross-sectional area were measured in the tonic soleus (SOL) and phasic tibialis anterior (TA). Cryostat sections were stained for alkaline phosphatase (ALP) activity to identify all capillaries, and proliferating cell nuclear antigen (PCNA) to localise the site of cellular proliferation. Cold-induced angiogenesis, indicated by an increase in capillary to fibre ratio (C:F), occurred in SOL of rats (approximately 20 % increase, P < 0.05) but not hamsters (approximately 9.5 % increase, n.s.), and in TA of hamsters (approximately 22 % increase, P < 0.01) but not rats (approximately 1 % increase, n.s.). The change in C:F was highest in the glycolytic cortex region of TA where fibre size is larger than in the oxidative core. Capillary-specific cell proliferation (co-localised ALP and PCNA labelling) increased in parallel with C:F. The total PCNA label density within the interstitium was some 5-fold higher than that co-localised with capillaries, but where angiogenesis occurred the relative increase in capillary labelling was 2-fold greater than for other cells of the interstitium. These data suggest a significant role for endothelial cell proliferation in the angiogenic response, indicative of the sprouting form of angiogenesis. There was a tendency for fibre hypertrophy in both SOL and TA of rats, especially in the core region of TA (P < 0.01), such that capillary density (CD) and intramuscular diffusion distances (DD) were largely unchanged following cold exposure. In contrast, fibre size was maintained in hamsters, DD reduced and CD increased compared to control TA (P < 0.01). In conclusion, cold acclimation stimulated angiogenesis in muscle of hamsters more than in rats, possibly due to a higher metabolic rate in the smaller species. Angiogenesis was also seen in SOL of rat, where oxidative capacity and muscle activity is higher than the TA. Thus, a combination of oxidative capacity, muscle activity, and fibre size may determine the degree of angiogenesis in response to low environmental temperature.

Muscle ischaemia in rats may be relieved by overload-induced angiogenesis.

Alleviation of muscle ischaemia by improving capillary supply has proved difficult, possibly reflecting the inability to substantially increase blood flow. We reasoned that muscle overload, which induces angiogenesis in the absence of altered blood flow, may be an alternative to drug therapy. Male Wistar rats underwent unilateral ligation of the common iliac artery, with or without ipsilateral extirpation of the tibialis anterior muscle. Six weeks later ischaemic (I) extensor digitorum longus (EDL) had a 10% (P < 0.05) decrease in relative muscle mass, while overloaded muscles (O) had undergone hypertrophy of 39% and 52% relative to contralateral (CL) and control (C) muscle masses, respectively (P < 0.01). Muscle atrophy was prevented by the combination of overload and ischaemia (O/I), with hypertrophy of 24% (vs. CL) and 35% (vs. C), respectively (P < 0.01). Changes in muscle fibre cross-sectional area paralleled the changes in muscle mass, with means of 1898 +/- 59, 1531 +/- 90, 2253 +/- 155 and 2292 +/- 80 mm2 for C, I, O and O/I, respectively (P < 0.01 vs. C and I). Capillary to fibre ratio (C:F) was significantly increased in overloaded (2.58 +/- 0.09) compared to contralateral (1.78 +/- 0.04), control (1.61 +/- 0.05) and ischaemic (1.73 +/- 0.06) muscles (P < 0.001). A similar increase in C:F was seen in overloaded plus ischaemic muscle (2.59 +/- 0.07) compared to contralateral (1.40 +/- 0.01) and control or ischaemic values (P < 0.01). In both O and O/I muscle groups, C:F and capillary density (CD) increased most in the region of EDL where fibre size was largest, while hypertrophy of fibres was least in the same region for both groups. These data suggest that the microvascular deficit evident in chronic muscle ischaemia may be alleviated by angiogenesis that is induced by mechanical stimuli via chronic muscle overload.

Chronic hypoxia induces prolonged angiogenesis in skeletal muscles of rat.

Skeletal muscle capillarity and fibre cross-sectional area were investigated within and between diaphragm (Diaph), extensor digitorum longus (EDL), soleus (SOL) and tibialis anterior (TA) muscles of control and chronic hypoxic (12 % O(2) for 6 weeks) adult male Wistar rats (final body mass approximately 355 g). Cryostat sections were stained for alkaline phosphatase activity to depict all capillaries, and for succinic dehydrogenase to demonstrate regional differences in oxidative capacity within the muscles. Hypoxia-induced angiogenesis occurred in all muscles (P < 0.01), with capillary-to-fibre ratio (C:F) being higher in the more active and oxidative muscles, Diaph (27 %) and SOL (26 %), than phasically active and glycolytic muscles, TA (21 %) and EDL (15 %). Diaph, SOL and EDL maintained fibre size, and hence showed an increased capillary density (CD) and reduced intramuscular diffusion distance (DD), whereas TA showed fibre hypertrophy and maintained CD and DD compared to control muscles. The extent of angiogenesis among different regions of muscle varied so as to suggest that muscle fibre size has an additional influence on capillary growth during chronic systemic hypoxia, which is progressive over an extended period of systemic hypoxia.

Relationship between capillary angiogenesis, fiber type, and fiber size in chronic systemic hypoxia.

Whether chronic hypoxia causes angiogenesis in skeletal muscle is controversial. Male Wistar rats, 5--6 wk of age, were kept at constant 12% O(2) for 3 wk, and frozen sections of their postural soleus (SOL), phasic extensor digitorum longus (EDL), and tibialis anterior (TA) muscles were compared with those of normoxic controls. Capillary supply increased in SOL muscles [capillary-to-fiber ratio (C/F) = 2.55 +/- 0.09 hypoxia vs. 2.17 +/- 0.06 normoxia; capillary density (CD) = 942 +/- 14 hypoxia vs. 832 +/- 20 mm(-2) normoxia, P < 0.01] but not in EDL muscles (C/F = 1.44 +/- 0.04 hypoxia vs. 1.42 +/- 0.04 normoxia; CD = 876 +/- 52 hypoxia vs. 896 +/- 24 mm(-2) normoxia). The predominantly glycolytic cortex of TA muscles showed higher C/F after hypoxia (1.79 +/- 0.09 vs. 1.53 +/- 0.05 normoxia, P < 0.05), whereas the mainly oxidative TA core with smaller fibers showed no change in capillarity. The region of the SOL muscle with large-sized (mean fiber area 2,843 +/- 128 microm(2)) oxidative fibers (90% type I) had a higher C/F (by 30%) and CD (by 25%), whereas there was no angiogenesis in the region with sparse (76%) and smaller-sized (2,200 +/- 85 microm(2)) type I fibers. Thus systemic hypoxia differentially induces angiogenesis between and within hindlimb skeletal muscles, with fiber size contributing either directly (via a metabolic stimulus) or indirectly (via a mechanical stimulus) to the process.

Differential effect of cold acclimation on blood composition in rats and hamsters.

Male rats and hamsters were exposed to a progressively lower air temperature and shorter photoperiod to simulate the onset of winter. Normothermic hamsters had a higher haematological oxygen carrying capacity (OCC) and coagulability (shorter prothrombin time and activated partial thromboplastin time) than rats. Cold acclimation significantly increased the OCC of rats, which parallels an increased metabolic rate, while no differences were observed in hamsters. Red cell transit time through filters was faster in the acclimated rats but not in hamsters, reflecting the lower mean cell volume due to a decreased rate of clearance from the circulation. Platelet counts were significantly lower in both cold-acclimated rats and hamsters, and there was a significant leucopenia in rats, which would reduce the degree of microvascular blockade. Whole blood viscosity, plasma viscosity, and serum osmolarity showed little change in either species. However, whole blood viscosity was significantly lower in cold-acclimated hamsters than control hamsters at the lowest shear rate tested (0.95 s(-1)). Interestingly, plasma viscosity and serum osmolarity were significantly lower in hamsters exposed to low temperatures for a shorter period (4 weeks), and may reflect the development of a reduced coagulability. These data suggest that blood composition in hamsters contributes to an innate tolerance of low temperatures, maintaining tissue perfusion under hypothermic conditions and aiding arousal from hibernation.

Development of the fluorescent microsphere technique for quantifying regional blood flow in small mammals.

We have demonstrated that the fluorescent microsphere technique can be used in small mammals for accurate determination of regional blood flows. In particular we have shown that 100% recovery of trapped microspheres is possible, that tissue digestion can be completed in a shorter time than previously reported, and the error-prone filtration method can be replaced with one of sedimentation. The method gave very good agreement among different fluorescent labels (r2 > 0.99) and low variability among tissues (mean coefficient of variation = 0.06). Simultaneous injection of radiolabelled and fluorescent microspheres established comparability between these methods (r2 = 0.96) for blood flows measured at rest, during vasodilator-induced hypotension, and in muscle hyperaemia during indirect electrical stimulation. Fluorescent microspheres can therefore replace radioactive microspheres for the determination of blood flow with advantages in both safety and cost, without loss of sensitivity.

Alterations in small arterioles precede changes in limb skeletal muscle after myocardial infarction.

We tested the hypothesis that alterations in arterioles in locomotor skeletal muscles in rats with myocardial infarction (MI), but before development of congestive heart failure (CHF), precede structural and functional changes commonly observed in limb muscle in association with CHF. Resting diameters of third- (A3) and fourth-order arterioles (A4) in extensor digitorum longus (EDL) muscle were significantly smaller in rats with nonfailing small and medium-sized MI compared with control animals. Dilation of A4 in response to 10(-4) M adenosine was significantly attenuated in both groups (P < 0.05), whereas dilation of A3 was unaltered. Microvessels from both groups of infarcted rats constricted to all doses of acetylcholine (10(-9), 10(-8), and 10(-7) M) and showed a significantly exaggerated vasoconstrictor response to norepinephrine (10(-9), 10(-8), and 10(-7) M) compared with microvessels in control rats (P < 0.05). Peak isometric tension of combined tibialis anterior and EDL muscles and muscle fatigue (final/peak tension x 100), measured during 5-min isometric supramaximal twitch contractions at 4 Hz, were similar in control and MI rats (218 +/- 7 vs. 213 +/- 15 g/g muscle and 52 +/- 1 vs. 51 +/- 9%, respectively; n = 5 for both). There was also no difference with respect to the proportion of oxidative fibers or capillary-to-fiber ratios. Our results indicate that, in rats with left ventricular dysfunction but without failure, decreased diameter and perturbations in reactivity of small arterioles precede alterations in skeletal muscle performance often seen at a later date in association with CHF. These findings are consistent with the notion of aberrant endothelial and smooth muscle function and may contribute to the maintenance of blood pressure after MI but before CHF.