Sebastian Albinsson

MicroRNAs Are Necessary for Vascular Smooth Muscle Growth, Differentiation, and Function

Objective—Regulation of vascular smooth muscle (VSM) proliferation and contractile differentiation is an important factor in vascular development and subsequent cardiovascular diseases. Recently, microRNAs (miRNAs) have been shown to regulate fundamental cellular processes in a number of cell types, but the integrated role of miRNAs in VSM in blood vessels is unknown. Here, we investigated the role of miRNAs in VSM by deleting the rate-limiting enzyme in miRNA synthesis, Dicer. Methods and Results—Deletion of Dicer in VSM results in late embryonic lethality at embryonic day 16 to 17, associated with extensive internal hemorrhage. The loss of VSM Dicer results in dilated, thin-walled blood vessels caused by a reduction in cellular proliferation. In addition, blood vessels from VSM-deleted Dicer mice exhibited impaired contractility because of a loss of contractile protein markers. We found this effect to be associated with a loss of actin stress fibers and partly rescued by overexpression of microRNA (miR)-145 or myocardin. Conclusion—Dicer-dependent miRNAs are important for VSM development and function by regulating proliferation and contractile differentiation.

Smooth Muscle miRNAs Are Critical for Post-Natal Regulation of Blood Pressure and Vascular Function

Phenotypic modulation of smooth muscle cells (SMCs) plays a key role in vascular disease, including atherosclerosis. Several transcription factors have been suggested to regulate phenotypic modulation of SMCs but the decisive mechanisms remain unknown. Recent reports suggest that specific microRNAs (miRNAs) are involved in SMC differentiation and vascular disease but the global role of miRNAs in postnatal vascular SMC has not been elucidated. Thus, the objective of this study was to identify the role of Dicer-dependent miRNAs for blood pressure regulation and vascular SMC contractile function and differentiation in vivo. Tamoxifen-inducible and SMC specific deletion of Dicer was achieved by Cre-Lox recombination. Deletion of Dicer resulted in a global loss of miRNAs in aortic SMC. Furthermore, Dicer-deficient mice exhibited a dramatic reduction in blood pressure due to significant loss of vascular contractile function and SMC contractile differentiation as well as vascular remodeling. Several of these results are consistent with our previous observations in SM-Dicer deficient embryos. Therefore, miRNAs are essential for maintaining blood pressure and contractile function in resistance vessels. Although the phenotype of miR-143/145 deficient mice resembles the loss of Dicer, the phenotypes of SM-Dicer KO mice were far more severe suggesting that additional miRNAs are involved in maintaining postnatal SMC differentiation.

Ticagrelor induces adenosine triphosphate release from human red blood cells.

RATIONALE The novel P2Y(12) antagonist ticagrelor inhibits ADP-induced platelet aggregation more rapidly and more potently than clopidogrel. Clinical trials have revealed dyspnea and asymptomatic ventricular pauses as side effects of ticagrelor. The mechanism behind these side effects is not known, but it is plausible that they are mediated by adenosine. OBJECTIVE Ticagrelor is known to increase adenosine concentrations by inhibiting red blood cell reuptake, but the potency of this effect may be too low to fully explain the adenosine related effects. The purpose of the present study was to determine whether ticagrelor has other effects on red blood cells (RBCs) that could contribute to explain the pleiotropic effects seen with ticagrelor treatment. METHODS AND RESULTS Using a luciferase-based bioluminescence assay, we studied ATP release in human blood. Human RBCs responded to ticagrelor in vitro by releasing substantial amounts of ATP in a dose-dependent manner (IC(50) 14μM). The rapid effect indicates release through membrane channels, which was supported by a depolarizing effect of ticagrelor and inhibition of ATP release by anion channel blockers. CONCLUSION In conclusion, our data show that, in vitro, ticagrelor can induce ATP release from human RBCs, which is subsequently degraded to adenosine. Further studies are warranted to determine what role this mechanism may play in the clinical effects of ticagrelor.

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.

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.

Mir-29 Repression in Bladder Outlet Obstruction Contributes to Matrix Remodeling and Altered Stiffness

Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.

Deletion of Dicer in Smooth Muscle Affects Voiding Pattern and Reduces Detrusor Contractility and Neuroeffector Transmission

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca2+ channels in the detrusor.

Knockout of the vascular endothelial glucocorticoid receptor abrogates dexamethasone-induced hypertension

Background Glucocorticoid-mediated hypertension is incompletely understood. Recent studies have suggested the primary mechanism of this form of hypertension may be through the effects of glucocorticoids on vascular tissues and not to excess sodium and water re-absorption as traditionally believed. Objective The goal of this study was to better understand the role of the vasculature in the generation and maintenance of glucocorticoid-mediated hypertension. Methods We created a mouse model with a tissue-specific knockout of the glucocorticoid receptor in the vascular endothelium. Results We show that these mice are relatively resistant to dexamethasone-induced hypertension. After 1 week of dexamethasone treatment, control animals have a mean blood pressure (BP) increase of 13.1 mmHg, whereas knockout animals have only a 2.7 mmHg increase (P < 0.001). Interestingly, the knockout mice have slightly elevated baseline BP compared with the controls (112.2 ± 2.5 vs. 104.6 ± 1.2 mmHg, P = 0.04), a finding which is not entirely explained by our data. Furthermore, we demonstrate that the knockout resistance arterioles have a decreased contractile response to dexamethasone with only 6.6% contraction in knockout vessels compared with 13.4% contraction in control vessels (P = 0.034). Finally, we show that in contrast to control animals, the knockout animals are able to recover a significant portion of their normal circadian BP rhythm, suggesting that the vascular endothelial glucocorticoid receptor may function as a peripheral circadian clock. Conclusion Our study highlights the importance of the vascular endothelial glucocorticoid receptor in several fundamental physiologic processes, namely BP homeostasis and circadian rhythm.

Reticulon 4 is necessary for endoplasmic reticulum tubulation, STIM1-Orai1 coupling, and store-operated calcium entry.

Background: Store-operated calcium entry requires the redistribution of ER localized STIM1 to ER-PM junctions. Results: Altering ER morphology by modulating reticulon expression affects STIM1 redistribution and consequently calcium entry. Conclusion: Reticulon shaping of the ER is critical for store-operated calcium entry. Significance: Understanding how membrane shaping proteins participate in the regulation of organelle function is essential in elucidating the structure-function interdependence of organelles. Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca2+ levels via SOCE and therefor are less susceptible to Ca2+ overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca2+ homeostasis.

Stretch-Sensitive Down-Regulation of the miR-144/451 Cluster in Vascular Smooth Muscle and Its Role in AMP-Activated Protein Kinase Signaling

Vascular smooth muscle cells are constantly exposed to mechanical force by the blood pressure, which is thought to regulate smooth muscle growth, differentiation and contractile function. We have previously shown that the expression of microRNAs (miRNAs), small non-coding RNAs, is essential for regulation of smooth muscle phenotype including stretch-dependent contractile differentiation. In this study, we have investigated the effect of mechanical stretch on miRNA expression and the role of stretch-sensitive miRNAs for intracellular signaling in smooth muscle. MiRNA array analysis, comparing miRNA levels in stretched versus non-stretched portal veins, revealed a dramatic decrease in the miR-144/451 cluster level. Because this miRNA cluster is predicted to target AMPK pathway components, we next examined activation of this pathway. Diminished miR-144/451 expression was inversely correlated with increased phosphorylation of AMPKα at Thr172 in stretched portal vein. Similar to the effect of stretch, contractile differentiation could be induced in non-stretched portal veins by the AMPK activator, AICAR. Transfection with miR-144/451 mimics reduced the protein expression level of mediators in the AMPK pathway including MO25α, AMPK and ACC. This effect also decreased AICAR-induced activation of the AMPK signaling pathway. In conclusion, our results suggest that stretch-induced activation of AMPK in vascular smooth muscle is in part regulated by reduced levels of miR-144/451 and that this effect may play a role in promoting contractile differentiation of smooth muscle cells.