PCSK9 activation promotes early atherosclerosis in a vascular microphysiological system.

Atherosclerosis is a primary precursor of cardiovascular disease (CVD), the leading cause of death worldwide. While proprotein convertase subtilisin/kexin 9 (PCSK9) contributes to CVD by degrading low-density lipoprotein receptors (LDLR) and altering lipid metabolism, PCSK9 also influences vascular inflammation, further promoting atherosclerosis. Here, we utilized a vascular microphysiological system to test the effect of PCSK9 activation or repression on the initiation of atherosclerosis and to screen the efficacy of a small molecule PCSK9 inhibitor. We have generated PCSK9 over-expressed (P+) or repressed (P-) human induced pluripotent stem cells (iPSCs) and further differentiated them to smooth muscle cells (viSMCs) or endothelial cells (viECs). Tissue-engineered blood vessels (TEBVs) made from P+ viSMCs and viECs resulted in increased monocyte adhesion compared to the wild type (WT) or P- equivalents when treated with enzyme-modified LDL (eLDL) and TNF-α. We also found significant viEC dysfunction, such as increased secretion of VCAM-1, TNF-α, and IL-6, in P+ viECs treated with eLDL and TNF-α. A small molecule compound, NYX-1492, that was originally designed to block PCSK9 binding with the LDLR was tested in TEBVs to determine its effect on lowering PCSK9-induced inflammation. The compound reduced monocyte adhesion in P+ TEBVs with evidence of lowering secretion of VCAM-1 and TNF-α. These results suggest that PCSK9 inhibition may decrease vascular inflammation in addition to lowering plasma LDL levels, enhancing its anti-atherosclerotic effects, particularly in patients with elevated chronic inflammation.

Variability in Heart Yield From Donation After Brain Death Between Organ Procurement Organizations: An Opportunity for Improvement.

Increasing the number of available hearts for transplantation is the best strategy to decrease waitlist mortality. This study examines organ procurement organizations (OPOs) and their role in the transplantation network to determine whether variability in performance exists across them. Adult deceased donors who met the criteria for brain death between 2010 and 2020 (inclusive) in the United States were examined. A regression model was fitted and internally validated using donor characteristics available at the time of organ recovery to predict the likelihood of heart transplantation. Subsequently, an expected heart yield was calculated for each donor using this model. Observed-to-expected (O/E) heart yield ratios for each OPO were calculated by dividing the number of hearts recovered for transplantation by the expected number of recoveries. There were 58 OPOs active during the study period, and on average, OPO activity grew over time. The mean O/E ratio among OPOs was 0.98 (standard deviation ± 0.18). Twenty-one OPOs consistently performed below the expected level (95% confidence intervals < 1.0) and generated a deficit of 1,088 expected transplantations during the study period. The proportion of hearts that were recovered for transplantation varied significantly by OPO categories: low tier 31.8%, mid tier 35.6%, and high tier 36.2% (p < 0.01), even as the expected yield was similar across tiers (p = 0.69). OPO performance accounts for 28% of the variability in successfully transplanting a heart after accounting for the role of referring hospitals, donor families, and transplantation centers. In conclusion, there is significant variability in volume and heart yield from brain-dead donors across OPOs.

Modeling early stage atherosclerosis in a primary human vascular microphysiological system.

Novel atherosclerosis models are needed to guide clinical therapy. Here, we report an in vitro model of early atherosclerosis by fabricating and perfusing multi-layer arteriole-scale human tissue-engineered blood vessels (TEBVs) by plastic compression. TEBVs maintain mechanical strength, vasoactivity, and nitric oxide (NO) production for at least 4 weeks. Perfusion of TEBVs at a physiological shear stress with enzyme-modified low-density-lipoprotein (eLDL) with or without TNFα promotes monocyte accumulation, reduces vasoactivity, alters NO production, which leads to endothelial cell activation, monocyte accumulation, foam cell formation and expression of pro-inflammatory cytokines. Removing eLDL leads to recovery of vasoactivity, but not loss of foam cells or recovery of permeability, while pretreatment with lovastatin or the P2Y11 inhibitor NF157 reduces monocyte accumulation and blocks foam cell formation. Perfusion with blood leads to increased monocyte adhesion. This atherosclerosis model can identify the role of drugs on specific vascular functions that cannot be assessed in vivo.

Application of Oxidative Stress to a Tissue-Engineered Vascular Aging Model Induces Endothelial Cell Senescence and Activation.

Clinical studies have established a connection between oxidative stress, aging, and atherogenesis. These factors contribute to senescence and inflammation in the endothelium and significant reductions in endothelium-dependent vasoreactivity in aged patients. Tissue-engineered blood vessels (TEBVs) recapitulate the structure and function of arteries and arterioles in vitro. We developed a TEBV model for vascular senescence and examined the relative influence of endothelial cell and smooth muscle cell senescence on vasoreactivity. Senescence was induced in 2D endothelial cell cultures and TEBVs by exposure to 100 µM H2O2 for one week to model chronic oxidative stress. H2O2 treatment significantly increased senescence in endothelial cells and mural cells, human neonatal dermal fibroblasts (hNDFs), as measured by increased p21 levels and reduced NOS3 expression. Although H2O2 treatment induced senescence in both the endothelial cells (ECs) and hNDFs, the functional effects on the vasculature were endothelium specific. Expression of the leukocyte adhesion molecule vascular cell adhesion molecule 1 (VCAM-1) was increased in the ECs, and endothelium-dependent vasodilation decreased. Vasoconstriction and endothelium-independent vasodilation were preserved despite mural cell senescence. The results suggest that the functional effects of vascular cell senescence are dominated by the endothelium.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower ß-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases ß-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of ß-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and ß-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that ß-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 µm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 µm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.

Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 µm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 µm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 µm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 µm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart.