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Mice are the most common animal model to investigate human disease and explore physiology. Mice are practical, cost efficient, and easily used for genetic manipulations. Although variability in cardiac structure and function among mouse strains is well noted, the effect of mouse strain on vascular stiffness indices is not known. Here, we compared mouse strain-dependent differences in key vascular stiffness indices among frequently used inbred mouse strains—C57Bl/6J, 129S, and Bl6/129S. In young healthy animals, baseline blood pressure and heart rate were identical in all strains, and independent of gender. However, both active in vivo and passive ex vivo vascular stiffness indices exhibited distinct differences. Specifically, both male and female 129S animals demonstrated the highest tensile stiffness, were least responsive to acetylcholine-induced vasorelaxation, and showed the lowest pulse wave velocity (PWV), an index of in vivo stiffness. C57Bl/6J mice demonstrated the highest PWV, lowest tensile stiffness, and the highest response to acetylcholine-induced vasorelaxation. Interestingly, within each strain, female mice had more compliant aortas. C57Bl/6J mice had thinner vessel walls with fewer layers, whereas 129S mice had the thickest walls with the most layers. Values in the Bl6/129S mixed background mice fell between C57Bl/6J and 129S mice. In conclusion, we show that underlying vascular properties of different inbred wild-type mouse strains are distinct, despite superficial similarities in blood pressure. For each genetic modification, care should be taken to identify proper controls, and conclusions might need to be verified in more than one strain to minimize the risk of false positive studies.

Endothelial dysfunction and arterial stiffening play major roles in cardiovascular diseases. The critical role for the miR-181 family in vascular inflammation has been documented. Here we tested whether the miR-181 family can influence the pathogenesis of hypertension and vascular stiffening. qPCR data showed a significant decrease in miR-181b expression in the aorta of the older mice. Eight miR-181a1/b1-/- mice and wild types (C57BL6J:WT) were followed weekly for pulse wave velocity (PWV) and blood pressure measurements. After 20 weeks, the mice were tested for endothelial function and aortic modulus. There was a progressive increase in PWV and higher systolic blood pressure in miR-181a1/b1-/- mice compared with WTs. At 21 weeks, aortic modulus was significantly greater in the miR-181a1/b1-/- group, and serum TGF-β was found to be elevated at this time. A luciferase reporter assay confirmed miR-181b targets TGF-βi (TGF-β induced) in the aortic VSMCs. In contrast, wire myography revealed unaltered endothelial function along with higher nitric oxide production in the miR-181a1/b1-/- group. Cultured VECs and VSMCs from the mouse aorta showed more secreted TGF-β in VSMCs of the miR-181a1/b1-/- group; whereas, no change was observed from VECs. Circulating levels of angiotensin II were similar in both groups. Treatment with losartan (0.6 g/L) prevented the increase in PWV, blood pressure, and vascular stiffness in miR-181a1/b1-/- mice. Immunohistochemistry and western blot for p-SMAD2/3 validated the inhibitory effect of losartan on TGF-β signaling in miR-181a1/b1-/- mice. Decreased miR-181b with aging plays a critical role in ECM remodeling by removing the brake on the TGF-β, pSMAD2/3 pathway.

The left ventricular ejection time is routinely measured from a peripheral arterial waveform. However, the arterial waveform undergoes constant transformation as the pulse wave propagates along the arterial tree. Our goal was to determine if the left ventricular ejection time measured peripherally in the arterial tree accurately reflected the ejection time measured through the aortic valve. Moreover, we examined/accessed the modulating influence of hemodynamics on ejection time measurements. Continuous wave Doppler waveform images through the aortic valve and the simultaneously obtained radial artery pressure waveforms were analyzed to determine central and peripheral ejection times, respectively. The peripheral ejection time was significantly longer than the simultaneously measured central ejection time (174.5±25.2 ms vs. 120.7±14.4 ms; P<0.0001; 17.4±8.7% increase). Moreover, the ejection time prolongation was accentuated at lower blood pressures, lower heart rate and lower pulse wave velocity. The time difference between centrally and peripherally measured ejection times likely reflects intrinsic vascular characteristics. Moreover, given that the ejection time also depends on blood pressure, heart rate and pulse wave velocity, peripherally measured ejection times might need to be adjusted to account for changes in these variables.

Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4 ⁻/⁻ mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430–460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor. Significance Non–image-forming opsins such as Opn4 regulate important physiological functions such as circadian photo-entrainment and affect. The recent discovery that melanopsin (Opn4) functions outside the central nervous system prompted us to explore a potential role for this receptor in blood vessel regulation. We hypothesized that Opn4-mediated signaling might explain the phenomenon of photorelaxation, for which a mechanism has remained elusive. We report the presence in blood vessels of Opn4 and demonstrate that it mediates wavelength-specific, light-dependent vascular relaxation. This photorelaxation signal transduction involves cGMP and phosphodiesterase 6, but not protein kinase G. Furthermore it is regulated by G protein-coupled receptor kinase 2 and involves vascular hyperpolarization. This receptor pathway can be harnessed for wavelength-specific light-based therapy in the treatment of diseases that involve altered vasoreactivity.

The lysyl oxidase family of enzymes (LOXs) catalyze oxidative deamination of lysine side chains on collagen and elastin to initialize cross-linking that is essential for the formation of the extracellular matrix (ECM). Elevated expression of LOXs is highly associated with diverse disease processes. To date, the inability to detect total LOX catalytic function in situ has limited the ability to fully elucidate the role of LOXs in pathobiological mechanisms. Using LOXL2 as a representative member of the LOX family, we developed an in situ activity assay by utilizing the strong reaction between hydrazide and aldehyde to label the LOX-catalyzed allysine (-CHO) residues with biotin-hydrazide. The biotinylated ECM proteins are then labeled via biotin-streptavidin interaction and detected by fluorescence microscopy. This assay detects the total LOX activity in situ for both overexpressed and endogenous LOXs in cells and tissue samples and can be used for studies of LOXs as therapeutic targets.To address the limitation of inability to detect lysyl oxidase enzymes (LOX) catalytic function associated with diverse disease processes, Wang et al. developed an in situ activity assay by utilizing the reactivity of hydrazides with imines and carbonyls. This assay detects the total LOX activity in situ for both overexpressed and endogenous LOXs in cells and tissue samples.

[...]while the anesthetic agents utilized are often used in non-PH patients, it is likely that they were titrated in a manner reflecting the disease burden. Indications for relocating the procedure to the hospital operating room included anticipated need for advanced hemodynamic monitoring, hemodynamic support beyond the scope of normal practice for an ambulatory surgical center, and postoperative hospital admission. Based on our results, we feel that our current practice is providing appropriate care to this patient population and that routine staffing of ophthalmology suites with cardiac anesthesiologists is not necessary.Funding Johns Hopkins Department of Anesthesiology and Critical Care Medicine 2019 intramural grant award for investigation of factors influencing perioperative outcomes in pulmonary hypertension patients.Conflicts of interest None.Individual author's contributions Dr. Sinead Nyhan: This author designed the project, collected the data, analyzed the data, and wrote the manuscript. Gender 57.1% female; 42.9% male Age, years (mean ± SD) 71.9 ± 10.6 years Primary diagnosis Pulmonary hypertension 100% Secondary diagnoses Hypertension 85.7% Heart failure with preserved ejection fraction/diastolic dysfunction 45.5% Diabetes 42.8% Hyperlipidemia 42.8% Obesity 37.1% Chronic kidney disease 28.5% Coronary artery disease 28.5% COPD 25.7% Valvulopathy 25.7% Heart failure with reduced ejection fraction 22.8% End stage renal disease 17.1% Obstructive sleep apnea 17.1% RV dysfunction 14.2% Cerebral vascular accident/transient ischemic attack 8.6% Sarcoidosis 8.6% Arrhythmia 5.7% Complete heart block 5.7% Hepatitis C 5.7% Interstitial lung disease 5.7% NSTEMI 5.7% Tobacco use (active) 5.7% Amyloidosis 2.9% CREST 2.9% Deep vein thrombosis 2.9% HIV 2.9% Pulmonary fibrosis 2.9% Restrictive lung disease 2.9% Scleroderma 2.9% Sickle cell disease 2.9% SLE 2.9% ASA I 0% II 2.9% III 48.6% IV 48.6% V 0% NYHA I 8.6% II 51.4% III 37.1% IV 2.9% V 0% Pulmonary hypertension Group (available in 6 patients) 1 66.6% 2 33.3% 3 0% 4 0% 5 0% Outpatient pulmonary hypertension medications (some patients were on a combination of multiple medications) Diuretics 54.2% Supplemental oxygen 17.1% Prostacyclin analogue 2.9% PDE5 inhibitors 17.1% Endothelin receptor antagonists 5.7% Steroids 5.7% Systolic blood pressure 140 ± 29.4 mmHg Diastolic blood pressure 70.6 ± 12.4 mmHg MAP 93.6 ± 15.5 mmHg Cardiac index (available in 8 patients) 2.3 ± 0.8 L/min/m2 LVEF 54.7 ± 14.4% RVSP 58.1 ± 10.6 mmHg PASP (available in 7 patients) 58.4 ± 6.5 mmHg PVR (available in 5 patients) 4.4 ± 2.2 Wood units RV dilation No 57.1% Yes 42.9% RV hypokinesis (available in 34 patients) No 79.4% Yes 20.6% TAPSE (available in 7 patients) 1.9 ± 1.0 cm 6MWD (available in 5 patients) 314.7 ± 188.7 m ProBNP (available in 15 patients) 870.4 ± 851.5 FVC/DLCO (available in 12 patients) 1.3 ± 0.7 Table 1 Baseline characteristics of 35 patients with pulmonary hypertension undergoing ophthalmologic procedures.a Surgical location Freestanding ambulatory surgical center 88.6% Tertiary academic center 11.4% Procedure (some patients received a combination of procedures) Lens implantation 42.8% Cataract extraction 34.2% Vitrectomy 17.1% Trabeculectomy 5.7% Keratoplasty 5.7% Retinal detachment repair 5.7% Conjunctival flap 2.9% Glaucoma implant 2.9% Corneal patch graft 2.9% Primary anesthesiologist Noncardiac 97.1%; Cardiac 2.9% Type of anesthesia (some cases were a combination of techniques) Sedation 71.4% Regional technique 62.9% Topical 54.3% General anesthesia with advanced airway 14.3% Regional or topical only 2.9% Intraoperative monitoring devices Arterial line 2.9% TEE 0% PA catheter 0% Noninvasive cardiac output monitor 0% Sedation medications (some patients received a combination of medications) Fentanyl 80% Midazolam 68% Propofol 48% Ketamine

Pulse wave velocity (PWV) has been recommended as an arterial damage assessment tool and a surrogate of arterial stiffness. However, the current technology does not allow to measure PWV both continuously and in real-time. We reported previously that peripherally measured ejection time (ET) overestimates ET measured centrally. This difference in ET is associated with the inherent vascular properties of the vessel. In the current study we examined ETs derived from plethysmography simultaneously at different peripheral locations and examined the influence of the underlying arterial properties on ET prolongation by changing the subject's position. We calculated the ET difference between two peripheral locations (ΔET) and its corresponding PWV for the same heartbeat. The ΔET increased with a corresponding decrease in PWV. The difference between ΔET in the supine and standing (which we call ET index) was higher in young subjects with low mean arterial pressure and low PWV. These results suggest that the difference in ET between two peripheral locations in the supine vs standing positions represents the underlying vascular properties. We propose ΔET in the supine position as a potential novel real-time continuous and non-invasive parameter of vascular properties, and the ET index as a potential non-invasive parameter of vascular reactivity.

Wire myography to test vasomotor functions of blood vessels ex‐vivo are well‐established for the systemic circulation, however, there is no consensus on protocols for pulmonary arteries. We created a standardized wire myography protocol for healthy rat PAs and validated this in a pulmonary hypertension (PH) model. Vessels stretched to higher initial tensions (5.0, 7.5 and 10.0 mN) exhibited a uniform response to phenylephrine, a larger dynamic range, and lower EC50 values. The endothelium‐mediated relaxation showed that moderate tensions (7.5 and 10.0 mN) produced robust responses with higher maximum relaxation and lower EC50 values. For endothelium independent responses, the higher initial tension groups had lower and more consistent EC50 values than the lower initial tension groups. Pulmonary arteries from rats with PH were more responsive to vasoactive drugs when subjected to a higher initial tension. Notably, vessels in the PH group subjected to 15.0 mN exhibited high dynamic ranges in contractile and relaxation responses without tearing. Lastly, we observed attenuated cholinergic responses in these vessels—consistent with endothelial dysfunction in PH. Therefore, a moderate initial tension of 7.5–10.0 mN is optimal for healthy rat pulmonary arteries and a higher initial tension of 15.0 mN is optimal for pulmonary arteries from animals with PH.

Tissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

Background The lower limit of cerebral autoregulation (LLA) refers to the mean blood pressure (BP) below which cerebral blood flow becomes pressure-dependent, resulting, among others, in an increased stroke risk. The LLA measured during cardiac surgery, correlates with the vascular measure Ambulatory Arterial Stiffness Index (AASI) determined from intraoperative continuous radial BP before cardiopulmonary bypass [ 1 ]. Using these data we investigated added factors that may enhance this correlation. Design and method The study population included 167 patients undergoing cardiac surgery (age 71 ± 8 years, 68% males) with good-quality BP records. The AASI. Additionally tested predictors were body-mass index (BMI), the coefficient of variation (SD/mean) of the systolic BP (SBP_CV), the composite variables BMI*(1-AASI), and its linear combination with SBP_CV. The odds ratio (OR) was determined by applying logistic regression to dichotomized predictors (by medians) and LLA- (by selected thresholds) adjusted to age, sex, diabetes mellitus, heart rate and preoperative diastolic BP. Results The Table shows that the LLA of individuals correlated significantly with each of the (continuous) predictors, and the adjusted OR increased for the composite predictors (dichotomized), while showing insensitivity to adjustors. The ORs reached a maximum for a LLA threshold of 55 mmHg. Conclusion The newly-defined composite predictors that increased the likelihood of predicting a LLA higher than 55 mmHg enhances our knowledge regarding the cerebral vasculature and autoregulation, and BP variability determinants of LLA under anesthesia. Table Univariate regression Adjusted OR Predictor r ( p -value) Mean [95% CI] ( p -value) AASI 0.27 (0.0004) 2.41 [1.16–5.00] (0.02) BMI −0.26 (0.0007) 3.77 [1.78–8.00] (0.0005) SBP_CV −0.29 (0.0002) 3.50 [1.71–7.16] (0.0006) BMI*(1-AASI) −0.35 (0.000005) 4.51 [2.16–9.40] (0.00006) 80*SBP_CV + BMI*(1-AASI) −0.44 (<0.000001) 8.20 [3.67–18.3] (<0.000001)