Karl Swärd

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Cholesterol Depletion Impairs Vascular Reactivity to Endothelin-1 by Reducing Store-Operated Ca2+ Entry Dependent on TRPC1

Abstract— The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-&bgr;-cyclodextrin (m&bgr;cd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ETA receptor) was reduced by m&bgr;cd and increased by cholesterol. Neither ligand binding nor colocalization of ETA and caveolin-1 was affected by m&bgr;cd. Ca2+ inflow via store-operated channels after depletion of intracellular Ca2+ stores was reduced in m&bgr;cd-treated caudal arteries, as shown by Mn2+ quench rate and intracellular [Ca2+] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase–polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by m&bgr;cd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni2+ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1–induced contractions were insensitive to the TRPC1 blocking antibody and to m&bgr;cd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to m&bgr;cd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1.

An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNFalpha up-regulates 5-HT(2A)-mediated contraction.

Asthma research is arguably limited by an absence of appropriate animal models to study the pharmacology of inflammatory mediators that affect airway hyperresponsiveness and remodelling. Here we assessed an assay based on mouse tracheal segments cultured for 1–32 days, and investigated contractile responses mediated by muscarinic and 5‐hydroxytryptamine (5‐HT) receptors following long‐term exposure to tumour necrosis factor‐alpha (TNFα). Following culture, in the absence of TNFα, maximum contractile responses to KCl and carbachol were similar, with an increase in response up to day two and a decrease to a stable level after 8 days. Maximal relaxations to isoprenaline were not affected by the culture procedure. The potency of KCl and isoprenaline increased throughout the study. DNA microarray data revealed that global gene expression changes were greater when tissues were introduced to culture than when they were maintained in culture. The morphology of smooth muscle cells was maintained throughout the culture period. 5‐HT induced a weak contraction in both fresh and cultured (up to 8 days) segments. Culture with TNFα produced a time‐ and concentration‐dependent increase in the maximal contraction to 5‐HT, evidently mediated by 5‐HT2A receptors, whereas, the potency for carbachol was reduced. In conclusion, the phenotype of airway smooth muscle remained largely intact during the culture period, even though minor changes were obtained during the first days of culture. The time‐dependent effect of TNFα indicates the importance of studying the long‐term effect of cytokines on the smooth muscle cells in relation to airway hyperresponsiveness and remodelling.

Cholesterol Dependence of Vascular ERK1/2 Activation and Growth in Response to Stretch: Role of Endothelin-1

Objective—Stretch-induced growth of the vascular wall plays a role in hypertension and neointima formation. Its signal pathways involve integrins, cytoskeleton, membrane receptors, and ion channels, some of which are organized in cholesterol-rich, membrane domains such as lipid rafts or caveolae. This study tested the role of rafts/caveolae in stretch-induced vascular growth by manipulation of membrane cholesterol contents. Methods and Results—Growth and protein synthesis were induced by mechanical stretch of rat portal veins in vitro. Sucrose gradient centrifugation showed stretch-induced tyrosine phosphorylation primarily in fractions containing caveolin-1. Disruption of membrane caveolae with use of methyl-&bgr;-cyclodextrin (m&bgr;cd) reduced weight gain, protein synthesis, and DNA synthesis to levels in unstretched, control veins. These effects were partially reversed by restoration of cellular cholesterol contents. Inhibited growth was associated with abolished activation of extracellular signal–regulated kinase (ERK) 1/2 in response to stretch and endothelin-1 (ET-1) but not to angiotensin II. Inhibition of ET-1 type A (ETA) receptors by RF139317 or endothelin-converting enzyme by phosphoramidone abolished stretch-induced ERK1/2 activation, which was, however, unaffected by removal of the endothelium. Conclusions—Stretch-induced growth signaling in vascular smooth muscle depends on cholesterol-rich, membrane microdomains by a mechanism involving ETA receptors that respond to endogenous ET-1 production.

The role of Caveolin-1 in cardiovascular regulation.

Caveolae are omega‐shaped membrane invaginations present in essentially all cell types in the cardiovascular system, and numerous functions have been ascribed to these structures. Caveolae formation depends on caveolins, cholesterol and polymerase I and transcript release factor‐Cavin (PTRF‐Cavin). The current review summarizes and critically discusses the cardiovascular phenotypes reported in caveolin‐1‐deficient mice. Major changes in the structure and function of heart, lung and blood vessels have been documented, suggesting that caveolae play a critical role at the interface between blood and surrounding tissue. According to an emerging paradigm, many of these changes are secondary to uncoupling of endothelial nitric oxide synthase. Thus, nitric oxide synthase not only synthesizes more nitric oxide in the absence of caveolin‐1, but also more superoxide with potential pathogenic consequences. It is further argued that the vasodilating drive from increased nitric oxide production in caveolin‐1‐deficient mice is balanced by changes in the vascular media that favour increased dynamic resistance regulation. Harnessing the therapeutic opportunities buried in caveolae, while challenging, could expand the arsenal of treatment options in cancer, lung disease and atherosclerosis.

MicroRNAs are essential for stretch-induced vascular smooth muscle contractile differentiation via miR-145-dependent expression of L-type calcium channels

Background: miRNAs regulate smooth muscle phenotype. Results: Deletion of miRNAs results in impaired stretch induction of contractile differentiation and reduced expression of L-type calcium channels. Conclusion: miRNAs are crucial for stretch-sensitive smooth muscle differentiation in part via miR-145-dependent expression of L-type calcium channels. Significance: These findings provide novel insights into the mechanism of smooth muscle phenotypic modulation in vascular disease. Stretch of the vascular wall is an important stimulus to maintain smooth muscle contractile differentiation that is known to depend on L-type calcium influx, Rho-activation, and actin polymerization. The role of microRNAs in this response was investigated using tamoxifen-inducible and smooth muscle-specific Dicer KO mice. In the absence of Dicer, which is required for microRNA maturation, smooth muscle microRNAs were completely ablated. Stretch-induced contractile differentiation and Rho-dependent cofilin-2 phosphorylation were dramatically reduced in Dicer KO vessels. On the other hand, acute stretch-sensitive growth signaling, which is independent of influx through L-type calcium channels, was not affected by Dicer KO. Contractile differentiation induced by the actin polymerizing agent jasplakinolide was not altered by deletion of Dicer, suggesting an effect upstream of actin polymerization. Basal and stretch-induced L-type calcium channel expressions were both decreased in Dicer KO portal veins, and inhibition of L-type channels in control vessels mimicked the effects of Dicer deletion. Furthermore, inhibition of miR-145, a highly expressed microRNA in smooth muscle, resulted in a similar reduction of L-type calcium channel expression. This was abolished by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN93, suggesting that Ca2+/calmodulin-dependent protein kinase IIδ, a target of miR-145 and up-regulated in Dicer KO, plays a role in the regulation of L-type channel expression. These results show that microRNAs play a crucial role in stretch-induced contractile differentiation in the vascular wall in part via miR-145-dependent regulation of L-type calcium channels.

Melatonin restores impaired contractility in aged guinea pig urinary bladder

Abstract:  Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin‐treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca2+]i) was estimated by epifluorescence microscopy of fura‐2‐loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca2+]i in response to the stimuli, suggesting a reduced Ca2+sensitivity. Indeed, the agonist‐induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca2+ sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI‐17 and MYPT1, and with the absence of CPI‐17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca2+ handling and Ca2+ sensitizations pathways. Moreover, the indoleamine restored age‐induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging‐related urinary bladder contractile impairment.

Influence of mitochondrial inhibition on global and local [Ca(2+)](I) in rat tail artery.

Inhibition of oxidative metabolism is often found to decrease contractility of systemic vascular smooth muscle, but not to reduce global [Ca2+]i. In the present study, we probe the hypothesis that it is associated with an altered pattern of intracellular Ca2+ oscillations (waves) influencing force development. In the rat tail artery, mitochondrial inhibitors (rotenone, antimycin A, and cyanide) reduced &agr;1-adrenoceptor–stimulated force by 50% to 80%, but did not reduce global [Ca2+]i. Less relaxation (about 30%) was observed after inhibition of myosin phosphatase activity with calyculin A, suggesting that part of the metabolic sensitivity involves the regulation of myosin 20-kDa light chain phosphorylation, although no decrease in phosphorylation was found in freeze-clamped tissue. Confocal imaging revealed that the mitochondrial inhibitors increased the frequency but reduced the amplitude of asynchronous cellular Ca2+ waves elicited by &agr;1 stimulation. The altered wave pattern, in association with increased basal [Ca2+]i, accounted for the unchanged global [Ca2+]i. Inhibition of glycolytic ATP production by arsenate caused similar effects on Ca2+ waves and global [Ca2+]i, developing gradually in parallel with decreased contractility. Inhibition of wave activity by the InsP3 receptor antagonist 2-APB correlated closely with relaxation. Furthermore, abolition of waves with thapsigargin in the presence of verapamil reduced force by about 50%, despite unaltered global [Ca2+]i, suggesting that contraction may at least partly depend on Ca2+ wave activity. This study therefore indicates that mitochondrial inhibition influences Ca2+ wave activity, possibly due to a close spatial relationship of mitochondria and the sarcoplasmic reticulum and that this contributes to metabolic vascular relaxation.

Ablation of SM22α decreases contractility and actin contents of mouse vascular smooth muscle

The actin‐binding protein SM22α marks contractile differentiation in smooth muscle, but its function is unknown. We tested its role in arterial contractility and stretch‐sensitive vascular protein synthesis. Active stress in depolarised mesenteric resistance arteries and portal veins was reduced by 40% in SM22α−/− mice. Passive and active arterial circumference–force relationships were shifted leftwards, whereas α1‐adrenergic responses were increased. Actin contents were 10–25% lower in vessels from SM22α−/− mice, but protein composition was otherwise similar. Synthesis of SM22α, calponin and α‐actin, but not β‐actin, was sensitive to stretch. Ablation of SM22α did not affect stretch sensitivity of any of these proteins. Thus, SM22α plays a role in contractility, possibly by affecting actin filament organisation.

Targeting smooth muscle microRNAs for therapeutic benefit in vascular disease.

In view of the bioinformatic projection that a third of all protein coding genes and essentially all biological pathways are under control of microRNAs (miRNAs), it is not surprising that this class of small RNAs plays roles in vascular disease progression. MiRNAs have been shown to be involved in cholesterol turnover, thrombosis, glucose homeostasis and vascular function. Some miRNAs appear to be specific for certain cells, and the role that such cell-specific miRNAs play in vascular disease is only beginning to be appreciated. A notable example is the miR-143/145 cluster which is enriched in mature and highly differentiated smooth muscle cells (SMCs). Here we outline and discuss the recent literature on SMC-expressed miRNAs in major vascular diseases, including atherosclerosis, neointima formation, aortic aneurysm formation, and pulmonary arterial hypertension. Forced expression of miR-145 emerges as a promising strategy for reduction and stabilization of atherosclerotic plaques as well as for reducing neointimal hyperplasia. It is concluded that if obstacles in the form of delivery and untoward effects of antimirs and mimics can be overcome, the outlook for targeting of SMC-specific miRNAs for therapeutic benefit in vascular disease is bright.