Genetically engineered mice for combinatorial cardiovascular optobiology

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Contribution of AGTR 1 Promoter Region Polymorphism to the Progression and Outcome of Sepsis in Patients with Various Comorbidities

Blood pressure dysregulation and circulatory failure are major contributors to the progression of sepsis and especially septic shock. One of the genes affecting the vascular endothelium and arteriolar tone is the angiotensin II receptor 1 gene (AGTR1). The AGTR1 rs275651 single-nucleotide polymorphism is associated with the development of angina, high altitude pulmonary edema, and hypertension. The significance of the AGTR1 rs275651 polymorphism in sepsis, particularly in patients with significant comorbidity, has not been studied previously.The aim of the study was to determine the impact of AGTR1 functional polymorphism on sepsis outcome in patients with various comorbidities, including cardiovascular disease and type 2 diabetes mellitus.Material and methods. A prospective study included 144 ICU patients of two clinical hospitals in Moscow, aged 18-75 years with clinical signs of sepsis (Sepsis-3, 2016).Results. In the group of patients with cardiovascular diseases, carriers of the TT AGTR1 rs275651 genotype had a lower mortality rate compared with carriers of the A allele (25 deaths out of 33 versus 16 out of 16, respectively, P=0.041, Fisher's exact test; P=0.0019, log-rank test). In the group of patients with diabetes mellitus (n=62), we also found significant differences in sepsis outcome based on the AGTR1 rs275651 genotype variant. The subgroup of TT AGTR1 rs275651 genotype carriers demonstrated significantly lower mortality compared with TA, AA genotypes carriers (27 deaths out of 41 and 20 out of 21, respectively, P=0.012, Fisher's exact test; OR=10.37; 95% CI: 1.26 to 85.5; P<0.0001, log-rank test).Conclusion. We found an association of the functional polymorphism AGTR1 -777 T>A (rs275651) with sepsis outcome in ICU patients with high-value baseline comorbidity: carriers of the more common TT genotype had lower mortality compared to carriers of the minor A allele.

Intravital Assessment of Pre-capillary Pulmonary Arterioles of Type-1 Diabetic Mice Shows Oxidative Damage and Increased Tone in Response to NOS Inhibition

Microvascular dilation, important for peripheral tissue glucose distribution, also modulates alveolar perfusion and is inhibited by loss of bioavailable nitric oxide (NO) in diabetes mellitus (DM). We hypothesized that DM-induced oxidative stress decreases bioavailable NO and pulmonary pre-capillary arteriolar diameter, causing endothelial injury. We examined sub-pleural pulmonary arterioles after acute NO synthase (NOS) inhibition with L-NAME in streptozotocin (STZ) and saline (CTRL)-treated C57BL/6J mice. Microvascular changes were assessed by intravital microscopy in the right lung of anesthetized mice with open-chest and ventilated lungs. Arteriolar tone in pulmonary arterioles (27.2 to 48.7 µm diameter), increased in CTRL mice (18.0 ± 11% constriction p=0.034, n=5) but decreased in STZ (13.6 ± 7.5% dilation p= 0.009, n=5), after L-NAME. Lung tissue DHE fluorescence (superoxide), inducible NOS expression, and protein nitrosylation (3-nitrotyrosine) increased in STZ mice and correlated with increased glucose levels (103.8 ± 8.8 mg/dL). Fluorescently-labeled fibrinogen administration and fibrinogen immunostaining showed fibrinogen adhesion, indicating endothelial injury in STZ mice. In CTRL mice, vasoconstriction to L-NAME was likely due to the loss of bioavailable NO. Vasodilation in STZ mice may be due to decreased formation of a vasoconstrictor or emergence of a vasodilator. These findings provide novel evidence that DM targets the pulmonary microcirculation and that decreased NO bioavailability and increased precapillary arteriolar tone could potentially lead to ventilation-perfusion abnormalities, exacerbating systemic DM complications.

Intraglomerular Dysfunction Predicts Kidney Failure in Type 2 Diabetes

No longitudinal data link intraglomerular hemodynamic dysfunction with end-stage kidney disease (ESKD) in people with type 2 diabetes (T2D). Afferent (R_A) and efferent (R_E) arteriolar tone and intraglomerular pressure (P_GLO) are not directly measurable in humans but are estimable from glomerular filtration rate (GFR), renal plasma flow (RPF), blood pressure, hematocrit, and plasma oncotic pressure. We examined the association of the R_A/R_E ratio and P_GLO with ESKD incidence in 237 Pima Indian persons with T2D who underwent serial measures of GFR (iothalamate) and RPF (<i>p</i>-aminohippurate). Their association with kidney structural lesions was also examined in a subset of 111 participants.<b> </b>Of the 237 participants (mean age 42 years, diabetes duration 11 years, GFR 153 ml/min, median ACR 36 mg/g), 69 progressed to ESKD during median follow-up of 17.5 years. In latent class analysis, distinct trajectories characterized by increasing R_A/R_E ratio (HR: 4.60, 95% CI 2.55-8.31) or elevated P_GLO followed by a rapid decline (HR: 2.96, 95% CI 1.45-6.02) strongly predicted incident ESKD. P_GLO(R²=21%, <i>p</i><0.0001) and R_A/R_E (R²=15%, <i>p</i><0.0001) also correlated with mesangial fractional volume, a structural predictor of DKD progression.<b> </b>In conclusion, intraglomerular hemodynamic parameters associated strongly with incident ESKD and correlated with structural lesions of DKD.

Intraglomerular Dysfunction Predicts Kidney Failure in Type 2 Diabetes

No longitudinal data link intraglomerular hemodynamic dysfunction with end-stage kidney disease (ESKD) in people with type 2 diabetes (T2D). Afferent (R_A) and efferent (R_E) arteriolar tone and intraglomerular pressure (P_GLO) are not directly measurable in humans but are estimable from glomerular filtration rate (GFR), renal plasma flow (RPF), blood pressure, hematocrit, and plasma oncotic pressure. We examined the association of the R_A/R_E ratio and P_GLO with ESKD incidence in 237 Pima Indian persons with T2D who underwent serial measures of GFR (iothalamate) and RPF (<i>p</i>-aminohippurate). Their association with kidney structural lesions was also examined in a subset of 111 participants.<b> </b>Of the 237 participants (mean age 42 years, diabetes duration 11 years, GFR 153 ml/min, median ACR 36 mg/g), 69 progressed to ESKD during median follow-up of 17.5 years. In latent class analysis, distinct trajectories characterized by increasing R_A/R_E ratio (HR: 4.60, 95% CI 2.55-8.31) or elevated P_GLO followed by a rapid decline (HR: 2.96, 95% CI 1.45-6.02) strongly predicted incident ESKD. P_GLO(R²=21%, <i>p</i><0.0001) and R_A/R_E (R²=15%, <i>p</i><0.0001) also correlated with mesangial fractional volume, a structural predictor of DKD progression.<b> </b>In conclusion, intraglomerular hemodynamic parameters associated strongly with incident ESKD and correlated with structural lesions of DKD.

Genetically Engineered Mice for Combinatorial Cardiovascular Optobiology

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase-mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in HCN4BAC-CatCh2/αMHC-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Decreased parenchymal arteriolar tone uncouples vessel-to-neuronal communication in a mouse model of vascular cognitive impairment

Chronic hypoperfusion is a key contributor to cognitive decline and neurodegenerative conditions, but the cellular mechanisms remain ill-defined. Using a multidisciplinary approach, we sought to elucidate chronic hypoperfusion-evoked functional changes at the neurovascular unit. We used bilateral common carotid artery stenosis (BCAS), a well-established model of vascular cognitive impairment, combined with an ex vivo preparation that allows pressurization of parenchymal arterioles in a brain slice. Our results demonstrate that mild (~ 30%), chronic hypoperfusion significantly altered the functional integrity of the cortical neurovascular unit. Although pial cerebral perfusion recovered over time, parenchymal arterioles progressively lost tone, exhibiting significant reductions by day 28 post-surgery. We provide supportive evidence for reduced adenosine 1 receptor-mediated vasoconstriction as a potential mechanism in the adaptive response underlying the reduced baseline tone in parenchymal arterioles. In addition, we show that in response to the neuromodulator adenosine, the action potential frequency of cortical pyramidal neurons was significantly reduced in all groups. However, a significant decrease in adenosine-induced hyperpolarization was observed in BCAS 14 days. At the microvascular level, constriction-induced inhibition of pyramidal neurons was significantly compromised in BCAS mice. Collectively, these results suggest that BCAS uncouples vessel-to-neuron communication—vasculo-neuronal coupling—a potential early event in cognitive decline.

Role of SOLUBLE GUANYLYL CYCLASE in RENAL AFFERENT AND EFFERENT ARTERIOLES

Renal arteriolar tone depends considerably on the dilatory action of nitric oxide (NO) via activation of soluble guanylyl cyclase (sGC) and cGMP action. NO deficiency and hypoxia/reoxygenation are important pathophysiological factors in the development of acute kidney injury. It is hypothesized that the NO-sGC-cGMP system functions differently in renal afferent (AA) compared to efferent arterioles (EA) and that the sGC activator cinaciguat differentially dilates these arterioles.Experiments were performed in isolated, perfused mouse glomerular arterioles. Hypoxia (0.1 % oxygen) was achieved by using a hypoxia chamber. Phosphodiesterase 5 (PDE5) and sGC subunits were considerably expressed on the mRNA level in AA. PDE5 inhibition with sildenafil, which blocks cGMP degradation, diminished the responses to Ang II bolus application in AA, but not significantly in EA. Vasodilation induced by sildenafil in Ang II-preconstricted vessels was stronger in EA than AA. Cinaciguat, an NO- and heme-independent sGC activator, dilated EA more strongly than AA after L-NAME (NO synthase inhibitor) treatment and preconstriction with Ang II. Cinaciguat-induced dilatation of L‑NAME pretreated and Ang II preconstricted arterioles was similar to controls without L-NAME treatment. Cinaciguat also induced dilatation in iodinated contrast medium treated AA. Further, it dilated EA, but not AA, after hypoxia/reoxygenation.The results reveal an important role of the NO-sGC-cGMP system for renal dilatation and that EA have a more potent sGC activated dilatory system. Further, AA seem to be more sensitive to hypoxia/reoxygenation than EA under these experimental conditions.

Comparison of Adrenergic and Purinergic Receptor Contributions to Vasomotor Responses in Mesenteric Arteries of C57BL/6J Mice and Wistar Rats

<b><i>Introduction:</i></b> The sympathetic nervous system can modulate arteriolar tone through release of adenosine triphosphate and norepinephrine, which bind to purinergic and adrenergic receptors (ARs), respectively. The expression pattern of these receptors, as well as the composition of neurotransmitters released from perivascular nerves (PVNs), can vary both in organ systems within and across species, such as mice and rats. <b><i>Objective:</i></b> This study explores the function of α_1A subtypes in mouse and rat third-order mesenteric arteries and investigates PVN-mediated vasoconstriction to identify which neurotransmitters are released from sympathetic PVNs. <b><i>Methods:</i></b> Third-order mesenteric arteries from male C57BL/6J mice and Wistar rats were isolated and mounted on a wire myograph for functional assessment. Arteries were exposed to phenylephrine (PE) and then incubated with either α_1A antagonist RS100329 (RS) or α_1D antagonist BMY7378, before reexposure to PE. Electrical field stimulation was performed by passing current through platinum electrodes positioned adjacent to arteries in the absence and presence of a nonspecific alpha AR blocker phentolamine and/or P2X₁-specific purinergic receptor blocker NF449. <b><i>Results:</i></b> Inhibition of α₁ ARs by RS revealed that PE-induced vasoconstriction is primarily mediated through α_1A and that the contribution of the α_1A AR is greater in rats than in mice. In the mouse model, sympathetic nerve-mediated vasoconstriction is mediated by both ARs and purinergic receptors, whereas in rats, vasoconstriction appeared to only be mediated by ARs and a nonpurinergic neurotransmitter. Further, neither model demonstrated that α_1D ARs play a significant role in PE-mediated vasoconstriction. <b><i>Conclusions:</i></b> The mesenteric arteries of male C57BL/6J mice and Wistar rats have subtle differences in the signaling mechanisms used to mediate vasoconstriction. As signaling pathways in humans under physiological and pathophysiological conditions become better defined, the current study may inform animal model selection for preclinical studies.

An update review of intradialytic hypotension: concept, risk factors, clinical implications and management

Intradialytic hypotension (IDH) is a frequent and serious complication of chronic haemodialysis, linked to adverse long-term outcomes including increased cardiovascular and all-cause mortality. IDH is the end result of the interaction between ultrafiltration rate (UFR), cardiac output and arteriolar tone. Thus excessive ultrafiltration may decrease the cardiac output, especially when compensatory mechanisms (heart rate, myocardial contractility, vascular tone and splanchnic flow shifts) fail to be optimally recruited. The repeated disruption of end-organ perfusion in IDH may lead to various adverse clinical outcomes affecting the heart, central nervous system, kidney and gastrointestinal system. Potential interventions to decrease the incidence or severity of IDH include optimization of the dialysis prescription (cool dialysate, UFR, sodium profiling and high-flux haemofiltration), interventions during the dialysis session (midodrine, mannitol, food intake, intradialytic exercise and intermittent pneumatic compression of the lower limbs) and interventions in the interdialysis period (lower interdialytic weight gain and blood pressure–lowering drugs). However, the evidence base for many of these interventions is thin and optimal prevention and management of IDH awaits further clinical investigation. Developing a consensus definition of IDH will facilitate clinical research. We review the most recent findings on risk factors, pathophysiology and management of IDH and, based on this, we call for a new consensus definition of IDH based on clinical outcomes and define a roadmap for IDH research.