The impact of temperature on vascular function in connection with vascular laser treatment.

Pulsed dye lasers are used effectively in the treatment of psoriasis with long remission time and limited side effects. It is, however, not completely understood which biological processes underlie its favorable outcome. Pulsed dye laser treatment at 585-595 nm targets hemoglobin in the blood, inducing local hyperthermia in surrounding blood vessels and adjacent tissues. While the impact of destructive temperatures on blood vessels has been well studied, the effects of lower temperatures on the function of several cell types within the blood vessel wall and its periphery are not known. The aim of our study is to assess the functionality of isolated blood vessels after exposure to moderate hyperthermia (45 to 60°C) by evaluating the function of endothelial cells, smooth muscle cells, and vascular nerves. We measured blood vessel functionality of rat mesenteric arteries (n=19) by measuring vascular contraction and relaxation before and after heating vessels in a wire myograph. To this end, we elicited vascular contraction by addition of either high potassium solution or the thromboxane analogue U46619 to stimulate smooth muscle cells, and electrical field stimulation (EFS) to stimulate nerves. For measurement of endothelium-dependent relaxation, we used methacholine. Each vessel was exposed to one temperature in the range of 45-60°C for 30 seconds and a relative change in functional response after hyperthermia was determined by comparison with the response per stimulus before heating. Non-linear regression was used to fit our dataset to obtain the temperature needed to reduce blood vessel function by 50% (Half maximal effective temperature, ET50). Our findings demonstrate a substantial decrease in relative functional response for all three cell types following exposure to 55°C-60°C. There was no significant difference between the ET50 values of the different cell types, which was between 55.9°C and 56.9°C (P>0.05). Our data show that blood vessel functionality decreases significantly when exposed to temperatures between 55°C-60°C for 30 seconds. The results show functionality of endothelial cells, smooth muscle cells, and vascular nerves is similarly impaired. These results help to understand the biological effects of hyperthermia and may aid in tailoring laser and light strategies for selective photothermolysis that contribute to disease modification of psoriasis after pulsed dye laser treatment.

Structure and function of resistance arteries from BB-creatine kinase and ubiquitous Mt-creatine kinase double knockout mice.

Increasing evidence indicates that the enzyme creatine kinase (CK) is intimately involved in microvascular contractility. The mitochondrial isoenzyme catalyses phosphocreatine synthesis from ATP, while cytoplasmic CK, predominantly the BB isoenzyme in vascular tissue, is tightly bound near myosin ATPase, where it favours ATP production from phosphocreatine to metabolically support vascular contractility. However, the effect of CK gene inactivation on microvascular function is hitherto unknown. We studied functional and structural parameters of mesenteric resistance arteries isolated from 5 adult male mice lacking cytoplasmic BB-CK and ubiquitous mitochondrial CK (CK-/-) vs 6 sex/age-matched controls. Using a Mulvany Halpern myograph, we assessed the acute maximum contractile force with 125 mM K+ and 10-5 M norepinephrine, and the effect of two inhibitors, dinitrofluorobenzene, which inhibits phosphotransfer enzymes (0.1 µM), and the specific adenylate kinase inhibitor P1, P5-di(adenosine 5') pentaphosphate (10-6 to 10-5 M). WT and CK-/- did not significantly differ in media thickness, vascular elasticity parameters, or acute maximum contractile force. CK-/- arteries displayed greater reduction in contractility after dinitrofluorobenzene 38%; vs 14% in WT; and after AK inhibition, 14% vs 5.5% in WT, and displayed abnormal mitochondria, with a partial loss of the inner membrane. Thus, CK-/- mice display a surprisingly mild phenotype in vascular dysfunction. However, the mitochondrial abnormalities and greater effect of inhibitors on contractility may reflect a compromised energy metabolism. In CK-/- mice, compensatory mechanisms salvage energy metabolism, as described for other CK knock-out models.

Sustained conduction of vasomotor responses in rat mesenteric arteries in a two-compartment in vitro set-up.

AIM: Conduction of vasomotor responses may contribute to long-term regulation of resistance artery function and structure. Most previous studies have addressed conduction of vasoactivity only during very brief stimulations. We developed a novel set-up that allows the local pharmacological stimulation of arteries in vitro for extended periods of time and studied the conduction of vasomotor responses in rat mesenteric arteries under those conditions. METHODS: The new in vitro set-up was based on the pressure myograph. The superfusion chamber was divided halfway along the vessel into two compartments, allowing an independent superfusion of the arterial segment in each compartment. Local and remote cumulative concentration-response curves were obtained for a range of vasoactive agents. Additional experiments were performed with the gap junction inhibitor 18ß-glycyrrhetinic acid and in absence of the endothelium. RESULTS: Phenylephrine-induced constriction and acetylcholine-induced dilation were conducted over a measured distance up to 2.84 mm, and this conduction was maintained for 5 minutes. Conduction of acetylcholine-induced dilation was inhibited by 18ß-glycyrrhetinic acid, and conduction of phenylephrine-induced constriction was abolished in absence of the endothelium. Constriction in response to high K+ was not conducted. Absence of remote stimulation dampened the local response to phenylephrine. CONCLUSION: This study demonstrates maintained conduction of vasoactive responses to physiological agonists in rat mesenteric small arteries likely via gap junctions and endothelial cells, providing a possible mechanism for the sustained functional and structural control of arterial networks.

Force-independent expression of c-fos mRNA by endothelin-1 in rat intact small mesenteric arteries.

AIM: Wall stress-independent signalling pathways were studied for endothelin-1 (ET-1)-induced c-fos expression in rat intact mesenteric small arteries. METHODS: Arteries were kept unmounted in Krebs buffer, equilibrated for 1 h and stimulated with vasoactive substances for 15-60 min. The c-fos mRNA expression was determined by real-time polymerase chain reaction. RESULTS: Stimulation with fetal bovine serum (FBS), phorbol 12-myristate 13-acetate (PMA) and ET-1 caused about a doubling of c-fos mRNA. The ET-1-induced c-fos expression was steady (15-60 min) and was inhibited by the inhibitor of the ET(A) receptor, BQ-123. Platelet-derived growth factor-B, angiotensin II and U46619 did not cause increased c-fos mRNA levels. The broad specificity inhibitor staurosporine inhibited the response to ET-1, but inhibitors of Rho-A kinase and phosphatidylinositol 3-kinase had no effect. However, inhibitors to tyrosine kinases, the MAP kinases [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase, p38], and to conventional protein kinase C showed no inhibition. Consistent with these findings, ET-1 did not cause activation of ERK1/2, a finding also seen in vessels held under pressure. In contrast, ET-1-induced c-fos expression was inhibited by the calcium chelator BAPTA, suggesting a role for intracellular calcium. This possibility was supported by the finding that raising the extracellular K(+) concentration caused increased expression of c-fos in a concentration-dependent manner. CONCLUSION: The results suggest that in the absence of wall stress, ET-1 is able to induce increased expression of c-fos independent of traditional growth pathways, such as MAP kinase. The mechanism appears to be calcium-dependent.

Differential structural adaptation to haemodynamics along single rat cremaster arterioles.

We tested the hypothesis that under physiological conditions, arterioles match their diameter to the level of shear stress. Haemodynamic and anatomical data were obtained in segments of the first-order arteriole of the rat cremaster muscle. Along this segment of ~10 mm in length, local blood pressure decreased from 68 +/- 4 mmHg upstream to 54 +/- 3 mmHg downstream (n = 5). Pulse pressure decreased from 8.2 +/- 1.3 mmHg upstream to 4.1 +/- 0.6 mmHg downstream. At the same locations, an increase in arteriolar diameter was measured in vivo, from 179 +/- 4 microm upstream to 203 +/- 4 microm downstream (n = 10). In vitro pressure-diameter relations of maximally dilated vessels showed that the passive diameter was larger in downstream than upstream segments over a 15-125 mmHg pressure range (n = 18). The wall stress was similar for the upstream vs. downstream location: 266 +/- 16 vs. 260 +/- 14 mN mm-2. However, shear stress decreased from 30 +/- 5 to 21 +/- 5 dyn cm-2 (3.0 +/- 0.5 to 2.1 +/- 0.5 N m-2; n = 4) along the artery. In conclusion, these results demonstrate that shear stress is not the only factor in determining vascular calibre. We suggest that arteriolar calibre may rather depend on an interplay between shear stress and the local pressure profile.