Mark S. Taylor

Endothelial calcium dysregulation underlies impairments in endothelial-dependent vasodilation (EDV), contributing to vascular disease progression. Repletion of 8-oxoguanine DNA glycosylase (OGG1), an enzyme involved in base excision repair, has been shown to forestall vascular disease progression. However, the role of OGG1 in regulating endothelial calcium dynamics and in preserving EDV is unknown. Here, calcium imaging via high-speed confocal microscopy and automated analytics was used to quantify the spatial and temporal parameters of endothelial calcium signals in the excised carotid arteries of male and female C57BL6J/FVBNJ mice aged 4–7 months with normal endogenous levels of OGG1, in mice lacking OGG1, and in mice with repleted human OGG1 targeted to the mitochondria. Mice lacking OGG1 exhibited an anomalous calcium phenotype characterized by a substantial increase in the basal tissue-wide frequency and spatial area of the endothelial calcium signals. Mitochondrial repletion of hOGG1 restored the calcium phenotype under unstimulated and acetylcholine-stimulated conditions. EDV was assessed using pressure myography. Mice lacking OGG1 exhibited significant impairments in EDV in response to acetylcholine, and the mitochondrial repletion of OGG1 rescued EDV. These findings highlight a novel role for OGG1 in endothelial signaling and suggest its importance in vascular homeostasis.

Background: Small conductance Ca²⁺ activated K⁺ channels (K_Ca2.3) are important regulators of vascular function. They provide Ca²⁺-dependent hyperpolarization of the endothelial membrane potential, promoting agonist-induced vasodilation. Another important mechanism of influence may occur through positive feedback regulation of endothelial Ca²⁺ signals, likely via amplification of influx through membrane cation channels. K_Ca2.3 channels have recently been implicated in flow-mediated dilation of the arterial vasculature and may contribute to the crucial homeostatic role of shear stress in preventing vascular wall remodeling and progressive vascular disease (i.e., atherosclerosis). The impact of K_Ca2.3 channels on endothelial Ca²⁺ signaling under physiologically relevant shear stress conditions remains unknown. Methods: In the current study, we employ mice expressing an endothelium-specific Ca²⁺ fluorophore (cdh5-GCaMP8) to characterize the K_Ca2.3 channel influence on the dynamic Ca²⁺ signaling profile along the arterial endothelium in the presence and absence of shear-stress. Results: Our data indicate K_Ca2.3 channels have a minimal influence on basal Ca²⁺ signaling in the carotid artery endothelium in the absence of flow, but they contribute substantially to amplification of Ca²⁺ dynamics in the presence of flow and their influence can be augmented through exogenous positive modulation. Conclusions: The findings suggest a pivotal role for K_Ca2.3 channels in adjusting the profile of homeostatic dynamic Ca²⁺ signals along the arterial intima under flow.

Endothelial dysfunction is a key feature of cardiovascular disease (CVD) including atherosclerosis. Impaired endothelial signaling leads to plaque formation, vascular wall remodeling and widespread cardiovascular dysregulation. The specific changes along the vascular intima associated with atherosclerosis, including the vulnerable circulation downstream of the flow obstruction, remain poorly understood. Previous findings from animal models suggest that preservation of a distinct Ca²⁺ signaling profile along the arterial endothelial network is crucial for maintaining vasculature homeostasis and preventing arterial disease. Ca²⁺ signaling in the intact human artery intima has not been well characterized. Here, we employed confocal imaging and a custom analysis algorithm to assess the spatially and temporally dynamic Ca²⁺ signaling profiles of human peripheral arteries isolated from the amputated legs of patients with advanced CVD (peripheral artery disease and/or diabetes) or patients who had lost limbs due to non-cardiovascular trauma. In all tibial artery branches (0.5–5 mm diameter) assessed, the intima consistently elicited a broad range of basal Ca²⁺ signals ranging from isolated focal transients to broad waves. Arteries from patients with existing CVD displayed a restricted intimal Ca²⁺ signaling pattern characterized by diminished event amplitude and area. Stimulation of type-4 vanilloid transient receptor potential channels (TRPV4) amplified endothelial Ca²⁺ signals; however, these signals remained smaller and spatially confined in arteries from patients with CVD verses those without CVD. Our findings reveal a characteristic underlying basal Ca²⁺ signaling pattern within the intima of human peripheral arteries and suggest a distinct truncation of the inherent Ca²⁺ profile with CVD.

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca²⁺) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K⁺) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca²⁺-activated K⁺ (K_Ca2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit K_Ca2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice were suppressed by CyPPA and, in the presence of CyPPA, oxytocin failed to augment contractions. In tissues exposed to oxytocin, depletion of internal Ca²⁺ stores with cyclopiazonic acid (CPA) impaired CyPPA relaxation, whereas blockade of nonselective cation channels (NSCC) using gadolinium (Gd³⁺) had no significant effect. Immunofluorescence revealed close proximity of K_Ca2.3 channels and ER inositol trisphosphate receptors (IP₃Rs) within myometrial smooth muscle cells. The findings suggest internal Ca²⁺ stores play a role in K_Ca2.3-dependent feedback control of uterine contraction and offer new insights for tocolytic therapies.

Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular homeostasis. Altered TRPV4 signaling has recently been implicated in various cardiovascular diseases, including hypertension and atherosclerosis. These versatile nonselective cation channels increase endothelial Ca²⁺ influx in response to various stimuli including shear stress and G protein-coupled receptor (GPCR) activation. Recent findings suggest TRPV4 channels produce localized Ca²⁺ transients at the endothelial cell plasma membrane that may allow targeted effector recruitment and promote large-scale Ca²⁺ events via release from internal stores (endoplasmic reticulum). However, the specific impact of TRPV4 channels on Ca²⁺ signaling in the intact arterial intima remains unknown. In the current study, we employ an endothelium-specific TRPV4 knockout mouse model (ecTRPV4^−/−) to identify and characterize TRPV4-dependent endothelial Ca²⁺ dynamics. We find that carotid arteries from both ecTRPV4^−/− and WT mice exhibit a range of basal and acetylcholine (ACh)-induced Ca²⁺ dynamics, similar in net frequency. Analysis of discrete Ca²⁺ event parameters (amplitude, duration, and spread) and event composite values reveals that while ecTRPV4^−/− artery endothelium predominantly produces large Ca²⁺ events comparable to and in excess of those produced by WT endothelium, they are deficient in a particular population of small events, under both basal and ACh-stimulated conditions. These findings support the concept that TRPV4 channels are responsible for generating a distinct population of focal Ca²⁺ transients in the intact arterial endothelium, likely underlying their essential role in vascular homeostasis.

Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling and atherosclerosis. Recent findings suggest that spatial and temporal tuning of local Ca²⁺ signals along the endothelium is vital to vascular function. In this study, we examined whether chronic flow disruption causes alteration of dynamic endothelial Ca²⁺ signal patterning associated with changes in vascular structure and function. For these studies, we performed surgical PL of the left carotid arteries of mice to establish chronic low flow for 2 weeks; right carotid arteries remained open and served as controls (C). Histological sections showed substantial remodeling of PL compared to C arteries, including formation of neointima. Isometric force measurements revealed increased PE‐induced contractions and decreased KCl‐induced contractions in PL vs C arteries. Endothelium‐dependent vasorelaxation in response to ACh; 10⁻⁸ to 10⁻⁵ mol/L) was significantly impaired in PL vs C vessels. Evaluation of endothelial Ca²⁺ using confocal imaging and custom analysis exposed distinct impairment of Ca²⁺ dynamics in PL arteries, characterized by reduction in active sites and truncation of events, corresponding to attenuated vasorelaxation. Our findings suggest that endothelial dysfunction in developing vascular disease may be characterized by distinct shifts in the spatial and temporal patterns of localized Ca²⁺ signals.

Rationale:Recent data from mesenteric and cerebral beds have revealed spatially restricted Ca2+ transients occurring along the vascular intima that control effector recruitment and vasodilation. Although Ca2+ is pivotal for coronary artery endothelial function, spatial and temporal regulation of functional Ca2+ signals in the coronary endothelium is poorly understood. Objective:We aimed to determine whether a discrete spatial and temporal profile of Ca2+ dynamics underlies endothelium-dependent relaxation of swine coronary arteries. Methods and Results:Using confocal imaging, custom automated image analysis, and myography, we show that the swine coronary artery endothelium generates discrete basal Ca2+ dynamics, including isolated transients and whole-cell propagating waves. These events are suppressed by depletion of internal stores or inhibition of inositol 1,4,5-trisphosphate receptors but not by inhibition of ryanodine receptors or removal of extracellular Ca2+. In vessel rings, inhibition of specific...

Although acute and chronic vasoregulation is inherently driven by endothelial Ca2+, control and targeting of Ca2+-dependent signals are poorly understood. Recent studies have revealed localized and dynamic endothelial Ca2+ events comprising an intricate signaling network along the vascular intima. Discrete Ca2+ transients emerging from both internal stores and plasmalemmal cation channels couple to specific membrane K+ channels, promoting endothelial hyperpolarization and vasodilation. The spatiotemporal tuning of these signals, rather than global Ca2+ elevation, appear to direct endothelial functions under physiologic conditions. In fact, altered patterns of dynamic Ca2+ signaling may underlie essential endothelial dysfunction in a variety of cardiovascular diseases. Advances in imaging approaches and analyses in recent years have allowed for detailed detection, quantification, and evaluation of Ca2+ dynamics in intact endothelium. Here, we discuss recent insights into these signals, including their sources of origination and their functional encoding. We also address key aspects of data acquisition and interpretation, including broad applications of automated high-content analysis.

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within...

Objective— Intermediate and small conductance K_Ca channels IK1 (K_Ca3.1) and SK3 (K_Ca2.3) are primary targets of endothelial Ca²⁺ signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca²⁺ transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1^−/−) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca²⁺ dynamics. Approach and Results— Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca²⁺ dynamics was not different between IK1^−/− and wild-type mice (P>0.05), the frequency of acetylcholine-stimulated (2 μmol/L) Ca²⁺ dynamics was greatly decreased in IK1^−/− endothelium (515±153 versus 1860±319 events; P−/−/SK3^T/T mice, ancillary suppression (+Dox) or overexpression (−Dox) of SK3 channels had little additional effect on the occurrence of events under basal or acetylcholine-stimulated conditions. However, SK3 overexpression did restore the decreased event amplitudes. Removal of extracellular Ca²⁺ reduced acetylcholine-induced Ca²⁺ dynamics to the same level in wild-type and IK1^−/− arteries. Blockade of IK1 and SK3 with the combination of charybdotoxin (0.1 μmol/L) and apamin (0.5 μmol/L) or transient receptor potential vanilloid 4 channels with HC-067047 (1 μmol/L) reduced acetylcholine Ca²⁺ dynamics in wild-type arteries to the level of IK1^−/−/SK3^T/T+Dox arteries. These drug effects were not additive. Conclusions— IK1, and to some extent SK3, channels exert a substantial positive feedback influence on endothelial Ca²⁺ dynamics.