Steffen-sebastian Bolz - Academia.edu (original) (raw)

Papers by Steffen-sebastian Bolz

Research paper thumbnail of Sphingosine-1-Phosphate Signaling Regulates Myogenic Responsiveness in Human Resistance Arteries

PLOS ONE, 2015

We recently identified sphingosine-1-phosphate (S1P) signaling and the cystic fibrosis transmembr... more We recently identified sphingosine-1-phosphate (S1P) signaling and the cystic fibrosis transmembrane conductance regulator (CFTR) as prominent regulators of myogenic responsiveness in rodent resistance arteries. However, since rodent models frequently exhibit limitations with respect to human applicability, translation is necessary to validate the relevance of this signaling network for clinical application. We therefore investigated the significance of these regulatory elements in human mesenteric and skeletal muscle resistance arteries. Mesenteric and skeletal muscle resistance arteries were isolated from patient tissue specimens collected during colonic or cardiac bypass surgery. Pressure myography assessments confirmed endothelial integrity, as well as stable phenylephrine and myogenic responses. Both human mesenteric and skeletal muscle resistance arteries (i) express critical S1P signaling elements, (ii) constrict in response to S1P and (iii) lose myogenic responsiveness following S1P receptor antagonism (JTE013). However, while human mesenteric arteries express CFTR, human skeletal muscle resistance arteries do not express detectable levels of CFTR protein. Consequently, modulating CFTR activity enhances myogenic responsiveness only in human mesenteric resistance arteries. We conclude that human mesenteric and skeletal muscle resistance arteries are a reliable and consistent model for translational studies. We demonstrate that the core elements of an S1P-dependent signaling network translate to human mesenteric resistance arteries. Clear species and vascular bed variations are evident, reinforcing the critical need for further translational study.

Research paper thumbnail of A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis

Cardiovascular Research, 2015

Retention of LDL cholesterol beneath the arterial endothelium initiates an inflammatory response ... more Retention of LDL cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real-time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less subendothelial LDL compared to wild-type littermates. We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.

Research paper thumbnail of The emerging role of Ca21 sensitivity regulation in promoting myogenic vasoconstriction

Growing evidence suggests that mechanisms which regulate the Ca 2þ sensitivity of the contractile... more Growing evidence suggests that mechanisms which regulate the Ca 2þ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca 2þ sensitivity is suited to partially uncouple intracellular Ca 2þ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca 2þ -dependent processes. Following a brief review of 'classical' Ca 2þ -dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca 2þ sensitivity. For the purpose of this review, Ca 2þ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin lightchain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca 2þ sensitivity will be discussed. Although Ca 2þ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca 2þ -dependent 'initiation/trigger' phase followed by a Ca 2þ -independent 'maintenance' phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca 2þ -dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca 2þ sensitivity, as an equal and complimentary partner of Ca 2þ -dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.

Research paper thumbnail of Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca21 changes and dilation in isolated resistance arteries

Using a novel vessel culture technique in combination with antisense oligonucleotide trans- fecti... more Using a novel vessel culture technique in combination with antisense oligonucleotide trans- fection, we tested whether the endothelium-derived hyperpolarizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arter- ies from hamster gracilis muscle (n519) were per- fused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in

Research paper thumbnail of Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity

PLOS ONE, 2015

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate... more The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTR ΔF508 ), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/ AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

Research paper thumbnail of The role of the sphingosine-1-phosphate signaling pathway in osteocyte mechanotransduction

Bone, 2015

Osteocytes are proposed to be the mechanosensory cells that translate mechanical loading into bio... more Osteocytes are proposed to be the mechanosensory cells that translate mechanical loading into biochemical signals during the process of bone adaptation. The lipid mediator sphingosine-1-phosphate (S1P) has been reported to play a role in the mechanotransduction process of blood vessels and also in the dynamic control of bone mineral homeostasis. Nevertheless, the potential role of S1P in bone mechanotransduction has yet to be elucidated. In this study, we hypothesized that a S1P cascade is involved in the activation of osteocytes in response to loading-induced oscillatory fluid flow (OFF) in bone. MLO-Y4 osteocyte-like cells express the necessary components of a functional S1P cascade. To examine the involvement of S1P signaling in osteocyte mechanotransduction, we applied OFF (1Pa, 1Hz) to osteocyte-like MLO-Y4 cells under conditions where the S1P signaling pathway was modulated. We found that decreased endogenous S1P levels significantly suppressed the OFF-induced intracellular calcium response. Addition of extracellular S1P to MLO-Y4 cells enhanced the synthesis and release of prostaglandin E2 (PGE2) under static cells and amplified OFF-induced PGE2 release. The stimulatory effect of OFF on the gene expression levels of osteoprotegerin (OPG) and receptor activator for nuclear factor κ B ligand (RANKL) was S1P dependent. Furthermore, the S1P2 receptor subtype was shown to be involved in OFF-induced PGE2 synthesis and release, as well as down-regulation of RANKL/OPG gene expression ratio. In summary, our data suggest that S1P cascade is involved in OFF-induced mechanotransduction in MLO-Y4 cells and that extracellular S1P exerts its effect partly through S1P2 receptors.

Research paper thumbnail of Tumor Necrosis Factor-α Underlies Loss of Cortical Dendritic Spine Density in a Mouse Model of Congestive Heart Failure

Journal of the American Heart Association, 2015

Heart failure (HF) is a progressive disorder characterized by reduced cardiac output and increase... more Heart failure (HF) is a progressive disorder characterized by reduced cardiac output and increased peripheral resistance, ultimately leading to tissue perfusion deficits and devastating consequences for several organs including the brain. We previously described a tumor necrosis factor-α (TNF-α)-dependent enhancement of posterior cerebral artery tone and concomitant reduced cerebral blood flow in a mouse model of early HF in which blood pressure remains minimally affected. HF is often associated with cognitive impairments such as memory deficits, even before any overt changes in brain structure and function occur. The pathophysiology underlying the development of cognitive impairments in HF is unknown, and appropriate treatment strategies are lacking. We used a well-established mouse model in which HF was induced by experimental myocardial infarction produced by permanent surgical ligation of the left anterior descending coronary artery (infarct size ≈25% of the left ventricular wal...

Research paper thumbnail of Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells

Molecular biology of the cell, Jan 15, 2015

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., a... more Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal ...

Research paper thumbnail of A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene

Proceedings of the National Academy of Sciences, 2009

Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congen... more Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congenital hereditary conditions in which there is a lack of cone function in humans cause achromatopsia, an autosomal recessive trait, characterized by low vision, photophobia, and lack of color discrimination. Herein we report the identification of mutations in the PDE6C gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase as a cause of autosomal recessive achromatopsia. Moreover, we show that the spontaneous mouse mutant cpfl1 that features a lack of cone function and rapid degeneration of the cone photoreceptors represents a homologous mouse model for PDE6C associated achromatopsia.

Research paper thumbnail of Elevation of plasma viscosity induces sustained NO-mediated dilation in the hamster cremaster microcirculation in vivo

Pfl�gers Archiv European Journal of Physiology, 1997

We studied whether a flow-independent increase of luminal wall shear stress (WSS) could dilate ha... more We studied whether a flow-independent increase of luminal wall shear stress (WSS) could dilate hamster arterioles in vivo and which endothelial mediators are potentially involved. To this end the plasma viscosity was elevated by exchanging blood for dextranerythrocyte solution thereby augmenting WSS. Diameters of small and large arterioles as well as red blood cell velocities were measured before and after exchange of blood for solutions of identical haematocrit containing either high-(HMWD) or low-molecular weight dextran (LMWD). The potential role of endothelial autacoids was investigated by local application of the NO-synthase inhibitor N G -nitro-L-arginine (L-NNA), the inhibitor of cyclooxygenase, indomethacin (3 µM), or the K + -channel blocker, tetrabutylammonium (TBA, 0.

Research paper thumbnail of A microfluidic platform for probing small artery structure and function

Lab on a Chip, 2010

Although pathologic changes to the structure and function of small blood vessels are hallmarks of... more Although pathologic changes to the structure and function of small blood vessels are hallmarks of various cardiovascular diseases, limitations of conventional investigation methods (i.e. pressure myography) have prohibited a comprehensive understanding of the underlying mechanisms. We developed a microfluidic device to facilitate assessment of resistance artery structure and function under physiological conditions (37 C, 45 mmHg transmural pressure). The platform allows for on-chip fixation, long-term culture and fully automated acquisition of up to ten dose-response sequences of intact mouse mesenteric artery segments (diameter z 250 micrometres and length z 1.5 mm) in a well-defined microenvironment. Even abluminal application of phenylephrine or acetylcholine (homogeneous condition) yielded dose-response relationships virtually identical to conventional myography. Unilateral application of phenylephrine (heterogeneous condition) limited constriction to the drug-exposed side, suggesting a lack of circumferential communication. The microfluidic platform allows us to address new fundamental biological questions, replaces a manually demanding procedure with a scalable approach and may enable organ-based screens to be routinely performed during drug development.

Research paper thumbnail of Myogenic effects enhance norepinephrine constriction: Inhibition by nitric oxide and felodipine

Kidney International, 1998

Myogenic effects enhance norepinephrine constriction: Inhibition by nitric oxide and felodipine. ... more Myogenic effects enhance norepinephrine constriction: Inhibition by nitric oxide and felodipine. Myogenic, pressure-induced vasoconstriction may amplify the effects of circulating vasoconstrictors. Through intravital microscopy in cremaster arterioles (31 to 115 m diameter), the relative contribution of myogenic responses (MR) to norepinephrine (NE)-induced constriction and the inhibitor potency of nitric oxide (NO) or a Ca 2ϩ entry blocker (CEB), felodipine (F), were examined. In 24 anesthetized hamsters, a vessel occluder was placed around the aorta to control cremaster vessel inflow pressure (IP). NE infusion increased blood pressure (by 50 Ϯ 2 mm Hg) and induced significant constriction (24% Ϯ 9%) in small arterioles (Ͻ 65 m) only. The constriction, which was not altered by adrenergic blockade, was dependent on the actual IP and was abolished when the IP increase was blocked. NO synthase (NOS) blockade unmasked a significant MR in large arterioles. F inhibited the MR predominantly in large vessels. In isolated microvessels, F completely blocked the pressure-induced Ca 2ϩ increase and MR. We conclude that circulating NE constricts muscle arterioles mainly by a myogenic mechanism. NO effectively opposes MR in larger arterioles, thus restricting MR and vasoconstrictor reinforcement to a small section of the vasculature being tightly controlled by metabolic signals. MR, which otherwise would impair adjustment of peripheral resistance, is reduced by CEB predominantly in larger arterioles, similar to NO.

Research paper thumbnail of Highly Effective Non-Viral Gene Transfer into Vascular Smooth Muscle Cells of Cultured Resistance Arteries Demonstrated by Genetic Inhibition of Sphingosine-1-Phosphate-Induced Vasoconstriction

Journal of Vascular Research, 2003

The linkage of vascular genes to specific functions will lead to a better understanding of cardio... more The linkage of vascular genes to specific functions will lead to a better understanding of cardiovascular pathophysiology. We developed an experimental model that enables the introduction of one or multiple gene(s) into vascular smooth muscle cells (VSMCs) of isolated resistance arteries. Exposure of the arteries to a green fluorescent protein (GFP)-encoding plasmid in combination with the transfectant Effectene((R)) for 20 h resulted in the expression of GFP in virtually all VSMCs in the arterial wall at fully preserved vascular function. For functional validation of the model, plasmids encoding the specific RhoA inhibitors C3 transferase or N19RhoA were transfected. In subsequent functional tests, inhibition of RhoA-dependent constriction induced by sphingosine-1-phosphate was similar to that in arteries treated with exogenous C3 transferase protein or the Rho kinase inhibitor Y27632. Responses to norepinephrine remained unaffected. This novel transfection technique enables gene function to be assessed in direct conjunction with signalling pathways in vascular tissue and provides, therefore, a new tool for microvascular proteomics.

Research paper thumbnail of Deafness and Stria Vascularis Defects in S1P2 Receptor-null Mice

Journal of Biological Chemistry, 2007

3 The abbreviations used are: S1P, sphingosine 1-phosphate; ABR, auditory brainstem response; DAP... more 3 The abbreviations used are: S1P, sphingosine 1-phosphate; ABR, auditory brainstem response; DAPI, 4Ј,6-diamidino-2-phenylindole; db, decibel; DPOAE, distortion product otoacoustic emission; SPL, sound pressure level; PBS, phosphate buffered saline; PFA, paraformaldehyde; MOPS, 4-morpholinepropanesulfonic acid.

Research paper thumbnail of Nitric Oxide Opposes Myogenic Pressure Responses Predominantly in Large Arterioles In Vivo

Hypertension, 1998

A myogenic vasoconstriction may amplify the effects of circulating vasoconstrictors. In cremaster... more A myogenic vasoconstriction may amplify the effects of circulating vasoconstrictors. In cremaster arterioles, the contribution of a myogenic component to the constriction on intravenous infusion of norepinephrine (NE) or angiotensin II (Ang II) was studied. Second, the role of endothelium-derived nitric oxide (NO) in the control of these myogenic constrictions and its site of action in the resistance vascular bed was investigated. In 30 anesthetized (pentobarbital) hamsters, the cremaster was prepared for intravital microscopy, and a pneumatic vessel occluder was placed around the aorta to vary blood pressure in the hindquarter of the animal. Intravenous infusion of NE (0.5 nmol/min) increased the systemic blood pressure by 52+/-2 mm Hg. Simultaneously, constrictions of up to 33+/-6% were observed in the small arterioles (SAs; maximal inner diameter, 36 to 65 microm). The constrictions were not significantly altered by a local adrenergic blockade but were abolished when the pressure elevation in the cremaster arterioles was blocked by partial occlusion of the abdominal aorta. Diameters in large arterioles (LAs; maximal inner diameter, 65 to 127 microm), however, did not change significantly on NE infusion. Similar responses in the arterioles were observed when the local pressure was increased stepwise from 60 to 120 mm Hg by partial opening of the aortic occluder. However, after treatment of the cremaster tissue with the inhibitor of the NO synthase, N(G)-nitro-L-arginine (L-NNA, 30 micromol/L), a significant pressure-induced constriction of up to 16+/-3% occurred in LAs, whereas the magnitude of the constriction in SAs remained unchanged. L-NNA also abolished the increases in blood flow that were observed with increments in pressure in control animals. Similar results were obtained when Ang II was used to increase blood pressure. We conclude that a myogenic constriction of SAs contributes markedly to the overall response of cremaster arterioles to circulating vasoconstrictors. NO effectively opposes the myogenic response in LAs, thus preventing myogenic constrictions in a vascular region where constriction cannot be fully controlled by metabolic dilation. If this attenuating effect of NO on myogenic constriction also takes place in other organs, it might be a decisive mechanism in controlling changes of total peripheral vascular resistance elicited by vasoconstrictors.

Research paper thumbnail of Impaired calmodulin binding of myosin-7A causes autosomal dominant hearing loss (DFNA11)

Human Mutation, 2004

Both myosin 7A (MYO7A) and calmodulin (CaM) are required for transduction and adaptation processe... more Both myosin 7A (MYO7A) and calmodulin (CaM) are required for transduction and adaptation processes in inner ear hair cells. We identified a novel heterozygous missense mutation ( c.2557C>T; p.R853C) in a family with autosomal dominant non-syndromic hearing loss that changes an evolutionarily invariant residue of the fifth IQ motif (IQ 5 ), a putative calmodulin (CaM) binding domain, of MYO7A. Functional effects of the p.R853C mutation were investigated in a physiological cellular environment by expressing MYO7A IQ 5 -containing peptides in smooth muscle cells of microarteries, in which overexpression of wildtype IQ 5 (with intact calmodulin binding) would be expected to compete with myosin light chain kinase (MLCK) for CaM binding. Indeed, analysis of calmodulin-dependent vasoconstriction suggests constitutive binding of CaM to the wildtype, but not the p.R853Cmutated IQ 5 motif at all physiologically relevant Ca 2+ concentrations. Thus our data suggest a disturbed CaM/MYO7A binding of the p.R853C mutant, this amino acid change may result in impaired adaptation to environmental stimuli and progressive deterioration of hearing transduction in heterozygotes. A defect in CaM/MYO7A interaction represents a novel pathomechanism for genetic hearing loss. It provides an attractive molecular target for therapeutic interventions aimed to delay or prevent the onset of hearing loss in families with mutations in myosin IQ domains.

Research paper thumbnail of Role of Sphingosine-1-Phosphate Phosphohydrolase 1 in the Regulation of Resistance Artery Tone

Circulation Research, 2008

Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is ... more Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S1P-degrading function, we hypothesized that S1P-phosphohydrolase 1 (SPP1) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP1. Overexpression of SPP1 (via transfection of a SPP1 wt ) reduced resting tone, Ca 2ϩ sensitivity, and myogenic vasoconstriction, whereas reduced SPP1 expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP1 (SPP1 H208A ) had no effect on any parameter tested, suggesting that catalytic activity of SPP1 is critical. The enhanced myogenic tone that follows overexpression of S1P-generating enzyme sphingosine kinase 1 (Sk1 wt ) was functionally antagonized by coexpression with SPP1 wt but not SPP1 H208A . SPP1 modulated vasoconstriction in response to 1 to 100 nmol/L exogenous S1P, a concentration range that was characterized as S1P 2 -dependent, based on the effect of S1P 2 inhibition by antisense oligonucleotides and 1 mol/L JTE013. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (1) restored S1P responses that were attenuated by SPP1 wt overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP1 is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk1 wt and S1P 2 receptor activation. SPP1 accesses extracellular S1P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP1 and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure. (Circ Res. 2008;103:315-324.)

Research paper thumbnail of Nitric Oxide-Induced Decrease in Calcium Sensitivity of Resistance Arteries Is Attributable to Activation of the Myosin Light Chain Phosphatase and Antagonized by the RhoA/Rho Kinase Pathway

Circulation, 2003

NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular s... more NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway. The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 micromol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 micromol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations. NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.

Research paper thumbnail of Cardioprotective and Vasodilatory Actions of Glucagon-Like Peptide 1 Receptor Are Mediated Through Both Glucagon-Like Peptide 1 Receptor-Dependent and -Independent Pathways

Circulation, 2008

Are Mediated Through Both Glucagon-Like Peptide 1 Receptor Dependent and Cardioprotective and Vas... more Are Mediated Through Both Glucagon-Like Peptide 1 Receptor Dependent and Cardioprotective and Vasodilatory Actions of Glucagon-Like Peptide 1 Receptor http://circ.ahajournals.org located on the World Wide Web at:

Research paper thumbnail of Oxidized LDL Increases the Sensitivity of the Contractile Apparatus in Isolated Resistance Arteries for Ca2+ via a Rho- and Rho Kinase-Dependent Mechanism

Circulation, 2000

Oxidized LDL reduces NO-mediated and endothelium-derived hyperpolarizing factor-mediated dilation... more Oxidized LDL reduces NO-mediated and endothelium-derived hyperpolarizing factor-mediated dilations. We studied, in hamster skeletal muscle resistance arteries (213+/-8 micrometer n=51), whether an altered vascular smooth muscle (VSM) response, particularly sensitization of the VSM contractile apparatus to Ca(2+), is involved in this oxLDL effect. Methods and Results-VSM or endothelial [Ca(2+)](i) and vascular diameter were measured in response to norepinephrine (0.3 micromol/L), sodium nitroprusside (10 micromol/L), C-type natriuretic peptide (1 to 100 nmol/L), papaverine (0.1 to 10 micromol/L), or the endothelial agonist acetylcholine (ACh, 0.01 to 1 micromol/L). OxLDL significantly increased resting VSM [Ca(2+)](i) (11+/-3%), decreased diameter (8+/-2%), and enhanced norepinephrine-induced constrictions. Dilations to sodium nitroprusside and C-type natriuretic peptide were significantly reduced (by 10+/-2% and 35+/-6%), whereas dose-response curves for papaverine and ACh were shifted to the right, despite unchanged increases in endothelial Ca(2+) after ACh. OxLDL significantly shifted the Ca(2+)-diameter relation to the left, as assessed by stepwise increasing extracellular Ca(2+) (0 to 3 mmol/L) in depolarized skeletal muscle resistance arteries. This sensitization to Ca(2+) by oxLDL was abolished after inhibition of Rho (C3 transferase) or Rho kinase (Y27632). OxLDL reduces VSM responsiveness to vasodilators by increasing VSM Ca(2+) but preferentially by sensitizing VSM to Ca(2+) via a Rho- and Rho kinase-dependent pathway.

Research paper thumbnail of Sphingosine-1-Phosphate Signaling Regulates Myogenic Responsiveness in Human Resistance Arteries

PLOS ONE, 2015

We recently identified sphingosine-1-phosphate (S1P) signaling and the cystic fibrosis transmembr... more We recently identified sphingosine-1-phosphate (S1P) signaling and the cystic fibrosis transmembrane conductance regulator (CFTR) as prominent regulators of myogenic responsiveness in rodent resistance arteries. However, since rodent models frequently exhibit limitations with respect to human applicability, translation is necessary to validate the relevance of this signaling network for clinical application. We therefore investigated the significance of these regulatory elements in human mesenteric and skeletal muscle resistance arteries. Mesenteric and skeletal muscle resistance arteries were isolated from patient tissue specimens collected during colonic or cardiac bypass surgery. Pressure myography assessments confirmed endothelial integrity, as well as stable phenylephrine and myogenic responses. Both human mesenteric and skeletal muscle resistance arteries (i) express critical S1P signaling elements, (ii) constrict in response to S1P and (iii) lose myogenic responsiveness following S1P receptor antagonism (JTE013). However, while human mesenteric arteries express CFTR, human skeletal muscle resistance arteries do not express detectable levels of CFTR protein. Consequently, modulating CFTR activity enhances myogenic responsiveness only in human mesenteric resistance arteries. We conclude that human mesenteric and skeletal muscle resistance arteries are a reliable and consistent model for translational studies. We demonstrate that the core elements of an S1P-dependent signaling network translate to human mesenteric resistance arteries. Clear species and vascular bed variations are evident, reinforcing the critical need for further translational study.

Research paper thumbnail of A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis

Cardiovascular Research, 2015

Retention of LDL cholesterol beneath the arterial endothelium initiates an inflammatory response ... more Retention of LDL cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real-time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less subendothelial LDL compared to wild-type littermates. We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.

Research paper thumbnail of The emerging role of Ca21 sensitivity regulation in promoting myogenic vasoconstriction

Growing evidence suggests that mechanisms which regulate the Ca 2þ sensitivity of the contractile... more Growing evidence suggests that mechanisms which regulate the Ca 2þ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca 2þ sensitivity is suited to partially uncouple intracellular Ca 2þ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca 2þ -dependent processes. Following a brief review of 'classical' Ca 2þ -dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca 2þ sensitivity. For the purpose of this review, Ca 2þ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin lightchain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca 2þ sensitivity will be discussed. Although Ca 2þ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca 2þ -dependent 'initiation/trigger' phase followed by a Ca 2þ -independent 'maintenance' phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca 2þ -dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca 2þ sensitivity, as an equal and complimentary partner of Ca 2þ -dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.

Research paper thumbnail of Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca21 changes and dilation in isolated resistance arteries

Using a novel vessel culture technique in combination with antisense oligonucleotide trans- fecti... more Using a novel vessel culture technique in combination with antisense oligonucleotide trans- fection, we tested whether the endothelium-derived hyperpolarizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arter- ies from hamster gracilis muscle (n519) were per- fused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in

Research paper thumbnail of Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity

PLOS ONE, 2015

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate... more The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTR ΔF508 ), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/ AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

Research paper thumbnail of The role of the sphingosine-1-phosphate signaling pathway in osteocyte mechanotransduction

Bone, 2015

Osteocytes are proposed to be the mechanosensory cells that translate mechanical loading into bio... more Osteocytes are proposed to be the mechanosensory cells that translate mechanical loading into biochemical signals during the process of bone adaptation. The lipid mediator sphingosine-1-phosphate (S1P) has been reported to play a role in the mechanotransduction process of blood vessels and also in the dynamic control of bone mineral homeostasis. Nevertheless, the potential role of S1P in bone mechanotransduction has yet to be elucidated. In this study, we hypothesized that a S1P cascade is involved in the activation of osteocytes in response to loading-induced oscillatory fluid flow (OFF) in bone. MLO-Y4 osteocyte-like cells express the necessary components of a functional S1P cascade. To examine the involvement of S1P signaling in osteocyte mechanotransduction, we applied OFF (1Pa, 1Hz) to osteocyte-like MLO-Y4 cells under conditions where the S1P signaling pathway was modulated. We found that decreased endogenous S1P levels significantly suppressed the OFF-induced intracellular calcium response. Addition of extracellular S1P to MLO-Y4 cells enhanced the synthesis and release of prostaglandin E2 (PGE2) under static cells and amplified OFF-induced PGE2 release. The stimulatory effect of OFF on the gene expression levels of osteoprotegerin (OPG) and receptor activator for nuclear factor κ B ligand (RANKL) was S1P dependent. Furthermore, the S1P2 receptor subtype was shown to be involved in OFF-induced PGE2 synthesis and release, as well as down-regulation of RANKL/OPG gene expression ratio. In summary, our data suggest that S1P cascade is involved in OFF-induced mechanotransduction in MLO-Y4 cells and that extracellular S1P exerts its effect partly through S1P2 receptors.

Research paper thumbnail of Tumor Necrosis Factor-α Underlies Loss of Cortical Dendritic Spine Density in a Mouse Model of Congestive Heart Failure

Journal of the American Heart Association, 2015

Heart failure (HF) is a progressive disorder characterized by reduced cardiac output and increase... more Heart failure (HF) is a progressive disorder characterized by reduced cardiac output and increased peripheral resistance, ultimately leading to tissue perfusion deficits and devastating consequences for several organs including the brain. We previously described a tumor necrosis factor-α (TNF-α)-dependent enhancement of posterior cerebral artery tone and concomitant reduced cerebral blood flow in a mouse model of early HF in which blood pressure remains minimally affected. HF is often associated with cognitive impairments such as memory deficits, even before any overt changes in brain structure and function occur. The pathophysiology underlying the development of cognitive impairments in HF is unknown, and appropriate treatment strategies are lacking. We used a well-established mouse model in which HF was induced by experimental myocardial infarction produced by permanent surgical ligation of the left anterior descending coronary artery (infarct size ≈25% of the left ventricular wal...

Research paper thumbnail of Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells

Molecular biology of the cell, Jan 15, 2015

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., a... more Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal ...

Research paper thumbnail of A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene

Proceedings of the National Academy of Sciences, 2009

Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congen... more Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congenital hereditary conditions in which there is a lack of cone function in humans cause achromatopsia, an autosomal recessive trait, characterized by low vision, photophobia, and lack of color discrimination. Herein we report the identification of mutations in the PDE6C gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase as a cause of autosomal recessive achromatopsia. Moreover, we show that the spontaneous mouse mutant cpfl1 that features a lack of cone function and rapid degeneration of the cone photoreceptors represents a homologous mouse model for PDE6C associated achromatopsia.

Research paper thumbnail of Elevation of plasma viscosity induces sustained NO-mediated dilation in the hamster cremaster microcirculation in vivo

Pfl�gers Archiv European Journal of Physiology, 1997

We studied whether a flow-independent increase of luminal wall shear stress (WSS) could dilate ha... more We studied whether a flow-independent increase of luminal wall shear stress (WSS) could dilate hamster arterioles in vivo and which endothelial mediators are potentially involved. To this end the plasma viscosity was elevated by exchanging blood for dextranerythrocyte solution thereby augmenting WSS. Diameters of small and large arterioles as well as red blood cell velocities were measured before and after exchange of blood for solutions of identical haematocrit containing either high-(HMWD) or low-molecular weight dextran (LMWD). The potential role of endothelial autacoids was investigated by local application of the NO-synthase inhibitor N G -nitro-L-arginine (L-NNA), the inhibitor of cyclooxygenase, indomethacin (3 µM), or the K + -channel blocker, tetrabutylammonium (TBA, 0.

Research paper thumbnail of A microfluidic platform for probing small artery structure and function

Lab on a Chip, 2010

Although pathologic changes to the structure and function of small blood vessels are hallmarks of... more Although pathologic changes to the structure and function of small blood vessels are hallmarks of various cardiovascular diseases, limitations of conventional investigation methods (i.e. pressure myography) have prohibited a comprehensive understanding of the underlying mechanisms. We developed a microfluidic device to facilitate assessment of resistance artery structure and function under physiological conditions (37 C, 45 mmHg transmural pressure). The platform allows for on-chip fixation, long-term culture and fully automated acquisition of up to ten dose-response sequences of intact mouse mesenteric artery segments (diameter z 250 micrometres and length z 1.5 mm) in a well-defined microenvironment. Even abluminal application of phenylephrine or acetylcholine (homogeneous condition) yielded dose-response relationships virtually identical to conventional myography. Unilateral application of phenylephrine (heterogeneous condition) limited constriction to the drug-exposed side, suggesting a lack of circumferential communication. The microfluidic platform allows us to address new fundamental biological questions, replaces a manually demanding procedure with a scalable approach and may enable organ-based screens to be routinely performed during drug development.

Research paper thumbnail of Myogenic effects enhance norepinephrine constriction: Inhibition by nitric oxide and felodipine

Kidney International, 1998

Myogenic effects enhance norepinephrine constriction: Inhibition by nitric oxide and felodipine. ... more Myogenic effects enhance norepinephrine constriction: Inhibition by nitric oxide and felodipine. Myogenic, pressure-induced vasoconstriction may amplify the effects of circulating vasoconstrictors. Through intravital microscopy in cremaster arterioles (31 to 115 m diameter), the relative contribution of myogenic responses (MR) to norepinephrine (NE)-induced constriction and the inhibitor potency of nitric oxide (NO) or a Ca 2ϩ entry blocker (CEB), felodipine (F), were examined. In 24 anesthetized hamsters, a vessel occluder was placed around the aorta to control cremaster vessel inflow pressure (IP). NE infusion increased blood pressure (by 50 Ϯ 2 mm Hg) and induced significant constriction (24% Ϯ 9%) in small arterioles (Ͻ 65 m) only. The constriction, which was not altered by adrenergic blockade, was dependent on the actual IP and was abolished when the IP increase was blocked. NO synthase (NOS) blockade unmasked a significant MR in large arterioles. F inhibited the MR predominantly in large vessels. In isolated microvessels, F completely blocked the pressure-induced Ca 2ϩ increase and MR. We conclude that circulating NE constricts muscle arterioles mainly by a myogenic mechanism. NO effectively opposes MR in larger arterioles, thus restricting MR and vasoconstrictor reinforcement to a small section of the vasculature being tightly controlled by metabolic signals. MR, which otherwise would impair adjustment of peripheral resistance, is reduced by CEB predominantly in larger arterioles, similar to NO.

Research paper thumbnail of Highly Effective Non-Viral Gene Transfer into Vascular Smooth Muscle Cells of Cultured Resistance Arteries Demonstrated by Genetic Inhibition of Sphingosine-1-Phosphate-Induced Vasoconstriction

Journal of Vascular Research, 2003

The linkage of vascular genes to specific functions will lead to a better understanding of cardio... more The linkage of vascular genes to specific functions will lead to a better understanding of cardiovascular pathophysiology. We developed an experimental model that enables the introduction of one or multiple gene(s) into vascular smooth muscle cells (VSMCs) of isolated resistance arteries. Exposure of the arteries to a green fluorescent protein (GFP)-encoding plasmid in combination with the transfectant Effectene((R)) for 20 h resulted in the expression of GFP in virtually all VSMCs in the arterial wall at fully preserved vascular function. For functional validation of the model, plasmids encoding the specific RhoA inhibitors C3 transferase or N19RhoA were transfected. In subsequent functional tests, inhibition of RhoA-dependent constriction induced by sphingosine-1-phosphate was similar to that in arteries treated with exogenous C3 transferase protein or the Rho kinase inhibitor Y27632. Responses to norepinephrine remained unaffected. This novel transfection technique enables gene function to be assessed in direct conjunction with signalling pathways in vascular tissue and provides, therefore, a new tool for microvascular proteomics.

Research paper thumbnail of Deafness and Stria Vascularis Defects in S1P2 Receptor-null Mice

Journal of Biological Chemistry, 2007

3 The abbreviations used are: S1P, sphingosine 1-phosphate; ABR, auditory brainstem response; DAP... more 3 The abbreviations used are: S1P, sphingosine 1-phosphate; ABR, auditory brainstem response; DAPI, 4Ј,6-diamidino-2-phenylindole; db, decibel; DPOAE, distortion product otoacoustic emission; SPL, sound pressure level; PBS, phosphate buffered saline; PFA, paraformaldehyde; MOPS, 4-morpholinepropanesulfonic acid.

Research paper thumbnail of Nitric Oxide Opposes Myogenic Pressure Responses Predominantly in Large Arterioles In Vivo

Hypertension, 1998

A myogenic vasoconstriction may amplify the effects of circulating vasoconstrictors. In cremaster... more A myogenic vasoconstriction may amplify the effects of circulating vasoconstrictors. In cremaster arterioles, the contribution of a myogenic component to the constriction on intravenous infusion of norepinephrine (NE) or angiotensin II (Ang II) was studied. Second, the role of endothelium-derived nitric oxide (NO) in the control of these myogenic constrictions and its site of action in the resistance vascular bed was investigated. In 30 anesthetized (pentobarbital) hamsters, the cremaster was prepared for intravital microscopy, and a pneumatic vessel occluder was placed around the aorta to vary blood pressure in the hindquarter of the animal. Intravenous infusion of NE (0.5 nmol/min) increased the systemic blood pressure by 52+/-2 mm Hg. Simultaneously, constrictions of up to 33+/-6% were observed in the small arterioles (SAs; maximal inner diameter, 36 to 65 microm). The constrictions were not significantly altered by a local adrenergic blockade but were abolished when the pressure elevation in the cremaster arterioles was blocked by partial occlusion of the abdominal aorta. Diameters in large arterioles (LAs; maximal inner diameter, 65 to 127 microm), however, did not change significantly on NE infusion. Similar responses in the arterioles were observed when the local pressure was increased stepwise from 60 to 120 mm Hg by partial opening of the aortic occluder. However, after treatment of the cremaster tissue with the inhibitor of the NO synthase, N(G)-nitro-L-arginine (L-NNA, 30 micromol/L), a significant pressure-induced constriction of up to 16+/-3% occurred in LAs, whereas the magnitude of the constriction in SAs remained unchanged. L-NNA also abolished the increases in blood flow that were observed with increments in pressure in control animals. Similar results were obtained when Ang II was used to increase blood pressure. We conclude that a myogenic constriction of SAs contributes markedly to the overall response of cremaster arterioles to circulating vasoconstrictors. NO effectively opposes the myogenic response in LAs, thus preventing myogenic constrictions in a vascular region where constriction cannot be fully controlled by metabolic dilation. If this attenuating effect of NO on myogenic constriction also takes place in other organs, it might be a decisive mechanism in controlling changes of total peripheral vascular resistance elicited by vasoconstrictors.

Research paper thumbnail of Impaired calmodulin binding of myosin-7A causes autosomal dominant hearing loss (DFNA11)

Human Mutation, 2004

Both myosin 7A (MYO7A) and calmodulin (CaM) are required for transduction and adaptation processe... more Both myosin 7A (MYO7A) and calmodulin (CaM) are required for transduction and adaptation processes in inner ear hair cells. We identified a novel heterozygous missense mutation ( c.2557C>T; p.R853C) in a family with autosomal dominant non-syndromic hearing loss that changes an evolutionarily invariant residue of the fifth IQ motif (IQ 5 ), a putative calmodulin (CaM) binding domain, of MYO7A. Functional effects of the p.R853C mutation were investigated in a physiological cellular environment by expressing MYO7A IQ 5 -containing peptides in smooth muscle cells of microarteries, in which overexpression of wildtype IQ 5 (with intact calmodulin binding) would be expected to compete with myosin light chain kinase (MLCK) for CaM binding. Indeed, analysis of calmodulin-dependent vasoconstriction suggests constitutive binding of CaM to the wildtype, but not the p.R853Cmutated IQ 5 motif at all physiologically relevant Ca 2+ concentrations. Thus our data suggest a disturbed CaM/MYO7A binding of the p.R853C mutant, this amino acid change may result in impaired adaptation to environmental stimuli and progressive deterioration of hearing transduction in heterozygotes. A defect in CaM/MYO7A interaction represents a novel pathomechanism for genetic hearing loss. It provides an attractive molecular target for therapeutic interventions aimed to delay or prevent the onset of hearing loss in families with mutations in myosin IQ domains.

Research paper thumbnail of Role of Sphingosine-1-Phosphate Phosphohydrolase 1 in the Regulation of Resistance Artery Tone

Circulation Research, 2008

Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is ... more Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S1P-degrading function, we hypothesized that S1P-phosphohydrolase 1 (SPP1) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP1. Overexpression of SPP1 (via transfection of a SPP1 wt ) reduced resting tone, Ca 2ϩ sensitivity, and myogenic vasoconstriction, whereas reduced SPP1 expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP1 (SPP1 H208A ) had no effect on any parameter tested, suggesting that catalytic activity of SPP1 is critical. The enhanced myogenic tone that follows overexpression of S1P-generating enzyme sphingosine kinase 1 (Sk1 wt ) was functionally antagonized by coexpression with SPP1 wt but not SPP1 H208A . SPP1 modulated vasoconstriction in response to 1 to 100 nmol/L exogenous S1P, a concentration range that was characterized as S1P 2 -dependent, based on the effect of S1P 2 inhibition by antisense oligonucleotides and 1 mol/L JTE013. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (1) restored S1P responses that were attenuated by SPP1 wt overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP1 is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk1 wt and S1P 2 receptor activation. SPP1 accesses extracellular S1P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP1 and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure. (Circ Res. 2008;103:315-324.)

Research paper thumbnail of Nitric Oxide-Induced Decrease in Calcium Sensitivity of Resistance Arteries Is Attributable to Activation of the Myosin Light Chain Phosphatase and Antagonized by the RhoA/Rho Kinase Pathway

Circulation, 2003

NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular s... more NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway. The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 micromol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 micromol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations. NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.

Research paper thumbnail of Cardioprotective and Vasodilatory Actions of Glucagon-Like Peptide 1 Receptor Are Mediated Through Both Glucagon-Like Peptide 1 Receptor-Dependent and -Independent Pathways

Circulation, 2008

Are Mediated Through Both Glucagon-Like Peptide 1 Receptor Dependent and Cardioprotective and Vas... more Are Mediated Through Both Glucagon-Like Peptide 1 Receptor Dependent and Cardioprotective and Vasodilatory Actions of Glucagon-Like Peptide 1 Receptor http://circ.ahajournals.org located on the World Wide Web at:

Research paper thumbnail of Oxidized LDL Increases the Sensitivity of the Contractile Apparatus in Isolated Resistance Arteries for Ca2+ via a Rho- and Rho Kinase-Dependent Mechanism

Circulation, 2000

Oxidized LDL reduces NO-mediated and endothelium-derived hyperpolarizing factor-mediated dilation... more Oxidized LDL reduces NO-mediated and endothelium-derived hyperpolarizing factor-mediated dilations. We studied, in hamster skeletal muscle resistance arteries (213+/-8 micrometer n=51), whether an altered vascular smooth muscle (VSM) response, particularly sensitization of the VSM contractile apparatus to Ca(2+), is involved in this oxLDL effect. Methods and Results-VSM or endothelial [Ca(2+)](i) and vascular diameter were measured in response to norepinephrine (0.3 micromol/L), sodium nitroprusside (10 micromol/L), C-type natriuretic peptide (1 to 100 nmol/L), papaverine (0.1 to 10 micromol/L), or the endothelial agonist acetylcholine (ACh, 0.01 to 1 micromol/L). OxLDL significantly increased resting VSM [Ca(2+)](i) (11+/-3%), decreased diameter (8+/-2%), and enhanced norepinephrine-induced constrictions. Dilations to sodium nitroprusside and C-type natriuretic peptide were significantly reduced (by 10+/-2% and 35+/-6%), whereas dose-response curves for papaverine and ACh were shifted to the right, despite unchanged increases in endothelial Ca(2+) after ACh. OxLDL significantly shifted the Ca(2+)-diameter relation to the left, as assessed by stepwise increasing extracellular Ca(2+) (0 to 3 mmol/L) in depolarized skeletal muscle resistance arteries. This sensitization to Ca(2+) by oxLDL was abolished after inhibition of Rho (C3 transferase) or Rho kinase (Y27632). OxLDL reduces VSM responsiveness to vasodilators by increasing VSM Ca(2+) but preferentially by sensitizing VSM to Ca(2+) via a Rho- and Rho kinase-dependent pathway.