Physical diminishes with ageing, but little is known of how the microvascular supply to skeletal muscle fibres is affected. To test the hypothesis that ageing alters blood flow control, we investigated network architecture and vasomotor responses of arterioles in the gluteus maximus muscle of young (2-3 months), adult (12-14 months) and old (18-20 months) C57BL6 male mice (n = 83) (Young, Adult and Old, respectively). Microvascular casts revealed that the total number, length and surface area of arteriolar segments (diameter, 10-50 µm) were not significantly different across age-groups. However, for arterioles with diameter of 30 µm, tortuosity and branch angles increased with age (P < 0.05). In anaesthetized mice, second-order (2A) distributing arterioles had similar resting (17 ± 1 µm) and maximal (37 ± 1 µm) diameters and similar responsiveness to cumulative (10 −10 −10 −4 M) superfusion of acetylcholine or phenylephrine. With superfusate oxygen level raised from 0 to 21%, 2A arteriolar constriction in Young (11 ± 1 µm) was greater (P < 0.05) than Adult and Old (5 ± 1 µm). Observed 1 mm upstream from microiontophoresis of ACh (1 µA, 1 s), conducted vasodilatation was 10 ± 1 µm in Young, 17 ± 1 µm in Adult and 6 ± 1 µm in Old (P < 0.05). With muscle contractions (2, 4 and 8 Hz; 30 s) arteriolar diameter increased similarly across age-groups (6 ± 1, 11 ± 1 and 18 ± 1 µm, respectively). Muscle mass and active tension were similar across age-groups yet postcontraction vasodilatation recovered more rapidly in Old versus Adult and Young (P < 0.05). With arteriolar network architecture maintained during ageing, the impairment in conducted vasodilatation and attenuation of postcontraction vasodilatation may compromise exercise tolerance.

Hypoxic pulmonary vasoconstriction (HVC), an intrinsic and assumed ubiquitous response of mammalian pulmonary blood vessels, matches regional ventilation to perfusion via an unknown O(2)-sensing mechanism. Global pulmonary hypoxia experienced by individuals suffering from chronic obstructive pulmonary disease or numerous hypoventilation syndromes, including sleep apnea, often produces maladaptive pulmonary hypertension, but pulmonary hypertension is not observed in diving mammals, where profound hypoxia is routine. Here we examined the response of cow and sea lion pulmonary arteries (PA) to hypoxia and observed the expected HVC in the former and a unique hypoxic vasodilation in resistance vessels in the latter. We then used this disparate response to examine the O(2)-sensing mechanism. In both animals, exogenous H(2)S mimicked the vasoactive effects of hypoxia in isolated PA. H(2)S-synthesizing enzymes, cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfur transferase, were identified in lung tissue from both animals by one-dimensional Western blot analysis and immunohistochemistry. The relationship between H(2)S production/consumption and O(2) was examined in real time by use of amperometric H(2)S and O(2) sensors. H(2)S was produced by sea lion and cow lung homogenate in the absence of O(2), but it was rapidly consumed when O(2) was present. Furthermore, consumption of exogenous H(2)S by cow lung homogenate, PA smooth muscle cells, and heart mitochondria was O(2) dependent and exhibited maximal sensitivity at physiologically relevant Po(2) levels. These studies show that HVC is not an intrinsic property of PA and provide further evidence for O(2)-dependent H(2)S metabolism in O(2) sensing.

Homocysteine (Hcy), a cardiovascular and neurovascular disease risk factor, is converted to hydrogen sulfide (H(2)S) through the transsulfuration pathway. H(2)S has attracted considerable attention in recent years for many positive effects on vascular health and homeostasis. Cystathionine β-synthase (CBS) is the first, and rate-limiting, enzyme in the transsulfuration pathway. Mutations in the CBS gene decrease enzymatic activity, which increases the plasma Hcy concentration, a condition called hyperhomocysteinemia (HHcy). Animal models of CBS deficiency have provided invaluable insights into the pathological effects of transsulfuration impairment and of both mild and severe HHcy. However, studies have also highlighted the complexity of HHcy and the need to explore the specific details of Hcy metabolism in addition to Hcy levels per se. There has been a relative paucity of work addressing the dysfunctional H(2)S production in CBS deficiency that may contribute to, or even create, HHcy-associated pathologies. Experiments using CBS knockout mice, both homozygous (-/-) and heterozygous (+/-), have provided 15 years of new knowledge and are the focus of this review. These murine models present the opportunity to study a specific mechanism for HHcy that matches one of the etiologies in many human patients. Therefore, the goal of this review was to integrate and highlight the critical information gained thus far from models of CBS deficiency and draw attention to critical gaps in knowledge, with particular emphasis on the modulation of H(2)S metabolism. We include findings from human and animal studies to identify important opportunities for future investigation that should be aimed at generating new basic and clinical understanding of the role of CBS and transsulfuration in cardiovascular and neurovascular disease.

The purpose of this study was to examine oxygen consumption (VO2) and heart rate kinetics during moderate and repeated bouts of heavy square-wave cycling from an exercising baseline. Eight healthy, male volunteers performed square-wave bouts of leg ergometry above and below the gas exchange threshold separated by recovery cycling at 35% VO2 peak. VO2 and heart rate kinetics were modeled, after removal of phase I data by use of a biphasic on-kinetics and monoexponential off-kinetics model. Fingertip capillary blood was sampled 45 s before each transition for base excess, HCO and lactate concentration, and pH. Base excess and HCO concentration were significantly lower, whereas lactate concentration and pH were not different before the second bout. The results confirm earlier reports of a smaller mean response time in the second heavy bout. This was the result of a significantly greater fast-component amplitude and smaller slow-component amplitude with invariant fast-component time constant. A role for local oxygen delivery limitation in heavy exercise transitions with unloaded but not moderate baselines is presented.

IntroductionMicrovessels are responsible for facilitating the exchange of nutrients between blood and tissue while maintaining a barrier against the undesired movement of other molecules. This barrier is most finely tuned and structurally competent at the interface between the blood and the brain, the blood-brain barrier (BBB). The BBB is formed by intercellular junctions that limit paracellular diffusion combined with a low-flux transcellular system that selectively transports molecules either into or out of the brain parenchyma. Increasing evidence implicates chronic BBB permeability in the onset of neurologic and neurovascular diseases (eg, Alzheimer disease, dementia, small-vessel disease, stroke). 1 Although the BBB prevents the flux of harmful substances into the brain, it can also exclude the delivery of therapeutic drugs. Understanding the molecular regulation of the BBB is therefore important for the understanding of disease etiology and treatment.Paracellular diffusion of molecules at the BBB is minimized by endothelial cell-cell (EC-EC) junctions. Chief among these are the adherens junctions and tight junctions, of which vascularendothelial cadherin (VE-cadherin; VEC) and claudin-5 are wellknown members, respectively. Although VEC is endothelial specific, the claudin family of proteins is found in many epithelial tissues. However, claudin-5 is specific to tight junctions of ECs and is required to block paracellular diffusion of small molecules (Ͻ ϳ 800 Da) in the microvasculature of the brain. 2 In particular, claudin-5 is now recognized as the predominant claudin isoform in tight junctions of the BBB. Although claudins 1, 10, 11, and 12 (at least) are also expressed in brain endothelium, claudin-5 mRNA is nearly 600-fold higher than the other claudins expressed in microvascular ECs freshly isolated from the brain. 3 On electron microscopy, plasma membranes of adjacent cells associate close enough to merge without apparent intercellular space at tight junctions. 4 Homocysteine (Hcy) is an intermediate aminothiol derived from methionine catabolism, and its elevation in plasma, known as hyperhomocysteinemia (HHcy), is a chronic condition associated with cerebral small-vessel disease, stroke, and dementia (Alzheimer and vascular). 5,6 The list of Hcy-mediated vasculopathies is still growing but includes endothelial dysfunction, vessel wall malformations, loss of extracellular matrix collagen, and loosening of the BBB in rodents and humans. 5,6 Mounting evidence suggests that an increase in BBB permeability is an important factor in initiating cerebral small-vessel disease and lacunar stroke. 7,8 Hence, identifying the mechanisms by which HHcy opens the BBB is of great clinical interest. We reported that treating human umbilical vein ECs with Hcy increases EC monolayer permeability and decreases expression of claudin-5. 9 However, the mechanism by which Hcy triggers these events has not been elucidated, and the intracellular mechanisms for decreased expression of claudin-5 in HHcy are unknown.Recently, a ...

Sweating threshold temperature and sweating sensitivity responses are measured to evaluate thermoregulatory control. However, analytic approaches vary, and no standardized methodology has been validated. This study validated a simple and standardized method, segmented linear regression (SReg), for determination of sweating threshold temperature and sensitivity. Archived data were extracted for analysis from studies in which local arm sweat rate (m(sw); ventilated dew-point temperature sensor) and esophageal temperature (T(es)) were measured under a variety of conditions. The relationship m(sw)/T(es) from 16 experiments was analyzed by seven experienced raters (Rater), using a variety of empirical methods, and compared against SReg for the determination of sweating threshold temperature and sweating sensitivity values. Individual interrater differences (n = 324 comparisons) and differences between Rater and SReg (n = 110 comparisons) were evaluated within the context of biologically important limits of magnitude (LOM) via a modified Bland-Altman approach. The average Rater and SReg outputs for threshold temperature and sensitivity were compared (n = 16) using inferential statistics. Rater employed a very diverse set of criteria to determine the sweating threshold temperature and sweating sensitivity for the 16 data sets, but interrater differences were within the LOM for 95% (threshold) and 73% (sensitivity) of observations, respectively. Differences between mean Rater and SReg were within the LOM 90% (threshold) and 83% (sensitivity) of the time, respectively. Rater and SReg were not different by conventional t-test (P > 0.05). SReg provides a simple, valid, and standardized way to determine sweating threshold temperature and sweating sensitivity values for thermoregulatory studies.

Objectives The mechanisms involved in activating pericytes, cells that ensheath capillaries, to engage in the formation of new capillaries, angiogenesis, remain unknown. In this study, the hypothesis was tested that pericytes could be stimulated to promote angiogenesis by driving the hypoxia-inducible factor (HIF) pathway. Methods Pericytes were stimulated with cobalt chloride (CoCl2) to activate the HIF pathway. Stimulated pericytes were co-cultured with endothelial cells in a wound healing assay and in a 3D collagen matrix assay of angiogenesis. A culture system of spinal cord tissue was used to assess microvascular outcomes after treatment with stimulated pericytes. Pharmaceutical inhibition of exosome production was also performed. Results Treatment with stimulated pericytes resulted in faster wound healing (1.92 ± 0.18 fold increase, p < 0.05), greater endothelial cord formation (2.9 ± 0.14 fold increase, p < 0.05) in cell culture assays and greater vascular density (1.78 ± 0.23 fold increase, p < 0.05) in spinal cord tissue. Exosome secretion and the physical presence of stimulated pericytes were necessary in the promotion of angiogenic outcomes. Conclusions These results elucidate a mechanism that may be exploited to enhance features of angiogenesis in the central nervous system (CNS).

Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-β-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.