Optimization of resting tension for wire myography in male rat pulmonary arteries.

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.

LOXL2 inhibition ameliorates pulmonary artery remodeling in pulmonary hypertension.

Background: Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. Methods and Results: Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen 5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. Conclusion: Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 is a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH. New & Noteworthy: Pulmonary arterial stiffening contributes to the progression of PAH and the deterioration of right heart function. This study shows that LOXL2 is upregulated in rat models of PH. LOXL2 inhibition halts pulmonary vascular remodeling and improves PA contractility, endothelial function and improves PA pressure, resulting in prolonged survival. Thus, LOXL2 is an important mediator of PA remodeling and stiffening in PH and a promising target to improve PA pressures and survival in PH.

Additional predictors of the lower limit of cerebral autoregulation during cardiac surgery.

OBJECTIVES: The lower limit of autoregulation (LLA) of cerebral blood flow was previously shown to vary directly with the Ambulatory Arterial Stiffness Index (AASI) redefined as 1-regression slope of DBP-versus-SBP readings invasively measured from the radial artery before the bypass. We aimed expanding the predictive capacity of the LLA with AASI by combining it with additional predictors and provide new indications whether mean arterial pressure (MAP) is above/below the LLA. DESIGN AND METHOD: In 181 patients undergoing cardiac surgery, mean (SD) age 71 (8) years), we identified from the demographic, preoperative and intraoperative characteristics independent and statistically significant 'single predictors' of the LLA (including AASI). This was achieved using multivariate linear regression with a backward-elimination technique. The single predictors combined with 1-AASI generated new multiplicative and additive composite predictors of the LLA. Indicators for the MAP-to-LLA difference (DIF) were determined using DIF-versus-predictor plots. The odds ratio (OR) for the DIF sign (Outcome = 1 for DIF≤0) and predictor-minus-median sign (Exposure = 1 for Predictor  ≤ Median) were calculated using logistic regression. RESULTS: BMI, 1-AASI and systolic coefficient of variation were identified single predictors that correlated similarly with the LLA ( r  = -0.26 to -0.27, P  < 0.001). The multiplicative and additive composite predictors displayed higher correlation with LLA ( r  = -0.41 and r  = -0.43, respectively, P  < 0.001) and improved LLA estimation. The adjusted OR for the composite predictors was nearly twice that of the single predictors. CONCLUSION: The novel composite predictors may enhance the LLA estimation and the ability to maintain MAP in the cerebral autoregulatory range during cardiac surgery.

Neonatal exposure to hypoxia induces early arterial stiffening via activation of lysyl oxidases.

Hypoxia in the neonatal period is associated with early manifestations of adverse cardiovascular health in adulthood including higher risk of hypertension and atherosclerosis. We hypothesize that this occurs due to activation of lysyl oxidases (LOXs) and the remodeling of the large conduit vessels, leading to early arterial stiffening. Newborn C57Bl/6 mice were exposed to hypoxia (FiO2  = 11.5%) from postnatal day 1 (P1) to postnatal day 11 (P11), followed by resumption of normoxia. Controls were maintained in normoxia. Using in vivo (pulse wave velocity; PWV) and ex vivo (tensile testing) arterial stiffness indexes, we determined that mice exposed to neonatal hypoxia had significantly higher arterial stiffness compared with normoxia controls by young adulthood (P60), and it increased further by P120. Echocardiography performed at P60 showed that mice exposed to hypoxia displayed a compensated dilated cardiomyopathy. Western blotting revelated that neonatal hypoxia accelerated age-related increase in LOXL2 protein expression in the aorta and elevated LOXL2 expression in the PA at P11 with a delayed decay toward normoxic controls. In the heart and lung, gene and protein expression of LOX/LOXL2 were upregulated at P11, with a delayed decay when compared to normoxic controls. Neonatal hypoxia results in a significant increase in arterial stiffness in early adulthood due to aberrant LOX/LOXL2 expression. This suggests an acceleration in the mechanical decline of the cardiovascular system, that contributes to increased risk of hypertension in young adults exposed to neonatal hypoxia that may increase susceptibility to further insults.

Transpulmonary amino acid metabolism in the sugen hypoxia model of pulmonary hypertension.

In pulmonary artery hypertension (PAH), emerging evidence suggests that metabolic abnormalities may be contributing to cellular dysfunction in PAH. Metabolic abnormalities such as glycolytic shift have been observed intracellularly in several cell types in PAH, including microvacular endothelial cells (MVECs). Concurrently, metabolomics of human PAH samples has also revealed a variety of metabolic abnormalities; however the relationship between the intracellular metabolic abnormalities and the serum metabolome in PAH remains under investigation. In this study, we utilize the sugen/hypoxia (SuHx) rodent model of PAH to examine the RV, LV and MVEC intracellular metabolome (using targeted metabolomics) in normoxic and SuHx rats. We additionally validate key findings from our metabolomics experiments with data obtained from cell culture of normoxic and SuHx MVECs, as well as metabolomics of human serum samples from two different PAH patient cohorts. Taken together, our data, spanning rat serum, human serum and primary isolated rat MVECs reveal that: (1) key classes of amino acids (specifically, branched chain amino acids-BCAA) are lower in the pre-capillary (i.e., RV) serum of SuHx rats (and humans); (2) intracellular amino acid levels (in particular BCAAs) are increased in SuHx-MVECs; (3) there may be secretion rather than utilization of amino acids across the pulmonary microvasculature in PAH and (4) an oxidized glutathione gradient is present across the pulmonary vasculature, suggesting a novel fate for increased glutamine uptake (i.e., as a source of glutathione). in MVECs in PAH. In summary, these data reveal new insight into the shifts in amino acid metabolism occurring across the pulmonary circulation in PAH.

Meeting Report: First Cardiovascular Outcomes Research in Perioperative Medicine Conference.

The first Cardiovascular Outcomes Research in Perioperative Medicine (COR-PM) conference took place on May 13, 2022, in Palm Springs, CA, and online. Here, we: (1) summarize the background, objective, and aims of the COR-PM meeting; (2) describe the conduct of the meeting; and (3) outline future directions for scientific meetings aimed at fostering high-quality clinical research in the broader perioperative medicine community.

Targeting LOXL2 Improves Arterial Stiffness and Function in Angiotensin II-induced Hypertension in Males but not Females.

Background: Hypertension accelerates arterial stiffening associated with natural aging. Aortic stiffness is both a cause and a consequence of isolated systolic hypertension. We identified lysyl oxidase-like 2 (LOXL2), a key matrix remodeling enzyme, as a potential therapeutic target for treating vascular stiffening. Here, we determine if LOXL2 depletion is protective against hypertension induced arterial stiffening, and we elucidate the sex differences present. Methods: Angiotensin II (Ang II) pumps were implanted in Loxl2 +/- and WT mice. Blood pressure and pulse wave velocity were measured noninvasively to assess hypertension and aortic stiffness. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and elastic properties. Histological analysis and Western blotting determined vascular wall properties. The effect of biomechanical strain on LOXL2 expression and cell alignment was determined via uniaxial cell stretching. Results: Ang II infusion induced hypertension in WT and Loxl2 +/- mice, and arterial stiffening was ameliorated in Loxl2 +/- male mice. LOXL2 depletion protected males from Ang II mediated potentiation of vasoconstriction, and attenuated passive arterial stiffening. Histological analysis showed increased aortic wall thickness and intralamellar distance with Ang II. Western blotting revealed an increase of LOXL2 accumulation and processing in hypertensive mice. Increased cyclic strain contributed to upregulation of LOXL2 in the aorta with induced hypertension. Conclusions: Arterial stiffening is increased with Ang II infusion; however, it is ameliorated in Loxl2 +/- male mice compared to WT despite developing Ang II-induced hypertension. This rise in arterial stiffness is driven by both VSMC response and matrix remodeling.

Selected Transesophageal Echocardiographic Parameters of Left Ventricular Diastolic Function Predict Length of Stay Following Coronary Artery Bypass Graft-A Prospective Observational Study.

(1) Importance: Abnormal left ventricular (LV) diastolic function, with or without a diagnosis of heart failure, is a common finding that can be easily diagnosed by intra-operative transesophageal echocardiography (TEE). The association of diastolic function with duration of hospital stay after coronary artery bypass (CAB) is unknown. (2) Objective: To determine if selected TEE parameters of diastolic dysfunction are associated with length of hospital stay after coronary artery bypass surgery (CAB). (3) Design: Prospective observational study. (4) Setting: A single tertiary academic medical center. (5) Participants: Patients with normal systolic function undergoing isolated CAB from September 2017 through June 2018. (6) Exposures: LV function during diastole, as assessed by intra-operative TEE prior to coronary revascularization. (7) Main Outcomes and Measures: The primary outcome was duration of postoperative hospital stay. Secondary intermediate outcomes included common postoperative cardiac, respiratory, and renal complications. (8) Results: The study included 176 participants (mean age 65.2 ± 9.2 years, 73% male); 105 (60.2%) had LV diastolic dysfunction based on selected TEE parameters. Median time to hospital discharge was significantly longer for subjects with selected parameters of diastolic dysfunction (9.1/IQR 6.6−13.5 days) than those with normal LV diastolic function (6.5/IAR 5.3−9.7 days) (p < 0.001). The probability of hospital discharge was 34% lower (HR 0.66/95% CI 0.47−0.93) for subjects with diastolic dysfunction based on selected TEE parameters, independent of potential confounders, including a baseline diagnosis of heart failure. There was a dose−response relation between severity of diastolic dysfunction and probability of discharge. LV diastolic dysfunction based on those selected TEE parameters was also associated with postoperative cardio-respiratory complications; however, these complications did not fully account for the relation between LV diastolic dysfunction and prolonged length of hospital stay. (9) Conclusions and Relevance: In patients with normal systolic function undergoing CAB, diastolic dysfunction based on selected TEE parameters is associated with prolonged duration of postoperative hospital stay. This association cannot be explained by baseline comorbidities or common post-operative complications. The diagnosis of diastolic dysfunction can be made by TEE.

Update on Perioperative Pediatric Pulmonary Hypertension Management.

Pediatric pulmonary hypertension is a disease that has many etiologies and can present anytime during childhood. Its newly revised hemodynamic definition follows that of adult pulmonary hypertension: a mean pulmonary artery pressure >20 mmHg. However, the pediatric definition stipulates that the elevated pressure must be present after the age of three months. The definition encompasses many different etiologies, and diagnosis often involves a combination of noninvasive and invasive testing. Treatment often is extrapolated from adult studies or based on expert opinion. Moreover, although general anesthesia may be required for pediatric patients with pulmonary hypertension, it poses certain risks. A thoughtful, multidisciplinary approach is needed to deliver excellent perioperative care.

How We Would Treat Our Own Pulmonary Hypertension if We Needed to Undergo Cardiac Surgery.

Pulmonary hypertension (PH) is a disease that has many etiologies and is particularly prevalent in patients presenting for cardiac surgery, with which it is linked to poor outcomes. This manuscript is intended to provide a comprehensive review of the impact of PH on the perioperative management of patients who are undergoing cardiac surgery. The diagnosis of PH often involves a combination of noninvasive and invasive testing, whereas preoperative optimization frequently necessitates the use of specific medications that affect anesthetic management of these patients. The authors postulate that a thoughtful, multidisciplinary approach is required to deliver excellent perioperative care. Furthermore, they use an index case to illustrate the implications of managing a patient with pulmonary hypertension who presents for cardiac surgery with cardiopulmonary bypass.

Probing tissue transglutaminase mediated vascular smooth muscle cell aging using a novel transamidation-deficient Tgm2-C277S mouse model.

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.

An in situ activity assay for lysyl oxidases.

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.

Pressure-based estimation of right ventricular ejection fraction: Validation as a clinically relevant target for drug development in a rodent model of pulmonary hypertension.

Depressed right ventricular ejection fraction (RVEF) has clear prognostic significance in patients with pulmonary arterial hypertension (PAH). Accordingly, improvements in RVEF represent a desirable end-point in the development of PAH therapies. However, current methods for determination of RVEF require measurement of RV volume and are relatively complex and costly. Here, we validate a novel method for quantitative estimation of RVEF in rats based entirely upon analysis of readily available RV pressure waveforms that eliminates the need for simultaneous volume measurement and can be rapidly applied. Right ventricular pressure and volume (conductance catheter) measurements acquired from 15 rats (7 controls, 8 sugen/hypoxia PAH; 220-250 g) were used for the study. Over the same 10 beat interval, RVEF was directly measured from the volume signal and estimated from the pressure signal. Simultaneous measures were compared by linear regression and Bland-Altman analysis to define bias (accuracy) and precision. Measured RVEF ranged from 0.19 to 0.60 (mean 0.44 ± 0.10) and estimated from 0.19 to 0.52 (mean 0.42 ± 0.09). Across the dataset there was strong correlation (r2 = 0.813), with minimal bias (0.01) and an overall error of 20% consistent with acceptable accuracy and precision. Study results support the potential utility of a method based entirely upon analysis of the RV pressure waveform for assessing drug effects on RVEF in rat models of PAH.

A novel approach to perioperative risk assessment for patients with pulmonary hypertension.

RATIONALE: Pulmonary hypertension (PH) is associated with significant perioperative morbidity and mortality. We hypothesised that pulmonary arterial hypertension (PAH) composite risk assessment scores could estimate perioperative risk for PH patients when adjusted for inherent procedural risk. METHODS: We identified patients in the Johns Hopkins PH Center Registry that had noncardiac surgery (including endoscopies) between September 2015 and January 2020. We collected information on preoperative patient-level and procedural variables and used logistic regression to evaluate associations with a composite outcome of death within 30 days or serious postoperative complication. We generated composite patient-level risk assessment scores for each subject and used logistic regression to estimate the association with adverse surgical outcomes. We adjusted multivariable models for inherent procedural risk of major cardiovascular events and used these models to generate a numerical PH perioperative risk (PHPR) score. RESULTS: Among 150 subjects, 19 (12.7%) reached the primary outcome, including 7 deaths (4.7%). Individual patient-level and procedural variables were associated with the primary outcome (all p<0.05). A composite patient-level risk assessment score built on three noninvasive parameters was strongly associated with reduced risk for poor outcomes (OR=0.4, p=0.03). This association was strengthened after adjusting the model for procedural risk. A PHPR score derived from the multivariable model stratified patients into low (0%), intermediate (≤10%), or high (>10%) risk of reaching the primary outcome. CONCLUSION: Composite PAH risk assessment scores can predict perioperative risk for PH patients after accounting for inherent procedural risk. Validation of the PHPR score in a multicentre, prospective cohort is warranted.

Red blood cell storage duration and peri-operative outcomes in paediatric cardiac surgery.

BACKGROUND: Prior research on red blood cell (RBC) storage duration and clinical outcomes in paediatric cardiac surgery has shown conflicting results. The purpose of this study was to evaluate whether blood stored for a longer duration is harmful in these patients. METHODS: We performed a retrospective cohort study of paediatric patients undergoing cardiac surgery at our institution between January 2011 and June 2015. Patients were stratified based on whether they were transfused RBCs stored for ≤15 days (fresher blood) or >15 days (older blood). The primary outcome was composite morbidity, with prolonged length of stay (LOS) as a secondary outcome. Subgroup analyses were performed after stratification by RBC transfusion volume (≤2 vs. >2 RBC units). Multivariable logistic regression models were used to assess the impact of RBC storage duration on composite morbidity and prolonged LOS. RESULTS: Of 461 patients, 122 (26·5%) received fresher blood and 339 (73·5%) received older blood. The overall rate of composite morbidity was 18·0% (n = 22) for patients receiving fresher blood and 13·6% (n = 46) for patients receiving older blood (P = 0·24). In the risk-adjusted model, patients receiving older blood did not exhibit an increased risk of composite morbidity (OR: 0·74, 95% CI: 0·37-1·47, P = 0·40) or prolonged LOS (OR: 0·72, 95% CI: 0·38-1·35, P = 0·30) compared to patients receiving fresher blood. Similar results were seen after stratification by RBC transfusion volume. CONCLUSIONS: Transfusing RBCs stored for a longer duration was not associated with an increased risk of morbidity or prolonged LOS in paediatric cardiac surgery patients.

Fast-Degrading Tissue-Engineered Vascular Grafts Lead to Increased Extracellular Matrix Cross-Linking Enzyme Expression.

Tissue-engineered vascular grafts (TEVGs) require adequate extracellular matrix (ECM) to withstand arterial pressure. Tissue transglutaminase (TG2) and lysyl oxidase (LOX) are enzymes that cross-link ECM proteins and play a pivotal role in the development of vascular stiffness associated with aging. The purpose of this study is to investigate the expression of ECM cross-linking enzymes and mechanisms of scaffold degeneration leading to vascular stiffness in TEVG remodeling. Fast- and slow-degrading electrospun TEVGs were fabricated using polydioxanone (PDO) and poly(L-lactide-co-caprolactone) (PLCL) copolymer, with a PDO/PLCL ratio of 9:1 for fast-degrading and 1:1 for slow-degrading graft. These grafts were implanted in rats (n = 5/group) as abdominal aortic interposition conduits. The grafts were harvested at 1 month to evaluate patency, mechanical properties, vascular neotissue formation, and the expression of ECM cross-linking enzymes. All TEVGs were patent without any aneurysmal formation at 1 month. ECM area, TG2-positive area, and LOX-positive area were significantly greater in fast-degrading TEVGs compared to slow-degrading TEVGs, with significantly less remaining scaffold. The mechanical properties of fast-degrading TEVGs were similar to that of native aorta, as demonstrated by strain-stress curve. In conclusion, at 1 month, fast-degrading TEVGs had rapid and well-organized ECM with greater TG2 and LOX expression and native-like mechanical properties, compared to slow-degrading TEVGs. Impact statement Around 1.4 million patients in the United States require arterial prostheses each year due to cardiovascular diseases. Current synthetic vascular grafts suffer from increased risk of infection, thrombosis, a lack of endothelialization, and compliance mismatch to the native vasculature. Tissue-engineered vascular graft (TEVGs) presented in this study exhibited tunable biodegradation profiles by controlling the polymer ratio of polydioxanone/poly(L-lactide-co-caprolactone). One month after implantation, the fast-degrading TEVGs exhibited mechanical properties similar to that of native aorta, formation of endothelium, and well-organized extracellular matrix (ECM) with increased expression of tissue transglutaminase and lysyl oxidases, which are critical to the ECM remodeling process.