Background Rheumatoid arthritis (RA) and atherosclerosis are chronic inflammatory diseases that share pathologic and molecular features. RA doubles the risk of cardiovascular disease (CVD) compared to the non-RA population. Common RA treatments are anti-inflammatory by design, but effects on CVD are unclear. Previously we reported the effect of SIR on IL6 and tumor necrosis factor (TNF) α signaling using a human endothelial cell (EC) and smooth muscle cell (SMC) co-culture system. Here we report additional analyses further characterizing effects of SIR on atherosclerotic (ATH) and oxidative (OX) stress pathways. Objectives To examine if RA drugs decrease ATH signaling and cellular stress in vascular cells under CVD conditions. Methods An in vitro surrogate system that co-cultures human ECs and SMCs was used to assess effects of RA drugs on vascular cells. Fluid flow conditions that drive cardiovascular health and CVD were applied. Atheroprone flow conditions were based on human hemodynamics from the carotid bifurcation, a site prone to developing atherosclerosis. The culture medium contained atherogenic risk factors including in vivo concentrations of oxidized LDL (oxLDL), soluble IL6 receptor (sIL6R), and TNF. Using RNA sequencing and microarray, we performed transcriptomic and biologic pathway analyses of surrogate system response. We compared treatments targeting pathogenic RA pathways, including anti-IL6 (SIR), anti-IL6 receptor (tocilizumab; TCZ), anti-TNF (adalimumab; ADA), and a small molecule JAK inhibitor (tofacitinib; TOF). The magnitude of pathway response was calculated as the L2 norm of the log2 fold change of genes in the pathway. Results The combination of atheroprone flow, sIL6R, TNF, and oxLDL (RA-CVD conditions) induced a robust response of inflammatory and OX stress pathways compared to healthy conditions (atheroprone flow without TNF and with non-oxLDL and sIL6R). The anti-IL6/IL6R treatments (SIR, TCZ) improved RA-CVD conditions by inhibiting key pathogenic pathways, including ATH signaling and NRF2-mediated OX stress. SIR attenuated the magnitude of RA-CVD response in ATH and OX stress pathways vs IgG or vehicle control the most: by 17% (adj P=0.035; Wilcoxon signed-rank test) and 34% (adj P=0.094) in ECs; and 49% (adj P=1.4e-5) and 47% (adj P=1.1e-3) in SMCs, respectively. TCZ was similar to SIR in restoring ECs and SMCs to healthy conditions in both pathways; ADA showed a weaker, similar trend compared to IL6 inhibition. TOF was not effective in suppressing (and tended to exacerbate) ATH and OX stress pathways in ECs. Conclusions IL6 pathway inhibitors SIR and TCZ potently suppressed ATH and cellular stress in vitro. The degree of suppression suggests that these drugs may mitigate the effects of atherogenic factors sIL6R, TNF, and oxLDL. In contrast, TNF inhibitor ADA was a less effective inhibitor of key CVD pathways, while JAK inhibitor TOF tended to exacerbate CVD pathways. Collectively, the data suggest that IL6 inhibition may provide more CVD benefit compared to RA drugs targeting other pathways. Acknowledgements Study sponsored by Janssen Research & Development, LLC, in collaboration with GlaxoSmithKline. Disclosure of Interest R. Feaver Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, S. Collado Employee of: HemoShear, LLC, S. Hoang Employee of: HemoShear, LLC, E. Berzin Employee of: HemoShear, LLC, A. Armstrong Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Gardner Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, H. Liu Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, A. Mackey Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Manka Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Shealy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, B. Blackman Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC

Objectives— The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. Approach and Results— We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. Conclusions— The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

Introduction: There is an urgent unmet need to improve the predictive value of animal and in vitro systems for drug development. The purpose of this study was to develop an in vitro vascular system that reflects the in vivo biology of the cynomolgus macaque (cyno). Methods: We co-cultured endothelial (EC) and smooth muscle cells (SMC) from cyno arteries and exposed them to physiologically relevant hemodynamics. Cyno velocity profiles were obtained from arterial regions prone to atherosclerosis (atheroprone) or healthy regions. Velocity waveforms were applied to the co-culture along with oxidized LDL from cyno or humans, and TNFα, which are pathogenic in humans and mimic advanced inflammatory conditions (AIC). Using RNA sequencing, we quantified the response of the system to 4 different statin treatments (atorvastatin, simvastatin, cerivastatin, rosuvastatin) under AIC. Using computational analyses of differentially expressed genes (DEGs), we characterized the response of our system by identifying functional biological themes that were associated with each statin treatment. Results: We found that AIC in the cyno system dramatically increased inflammatory DEGs and decreased vascular health DEGs. Cerivastatin elicited the most DEGs in both the cyno and human vascular system while the response to rosuvastatin did not reach statistical significance for any genes. Statin treatment decreased inflammatory cytokine gene expression, but not adhesion molecule expression under AIC. Except for rosuvastatin, all statins tested significantly increased the expression of genes that promote vascular health, while suppressing the expression of inflammatory genes. Biological themes were regulated by statin treatment in both the human and cyno vascular systems including: cholesterol biosynthesis, thrombosis, ethanol degradation, cell cycle regulation and notch signaling. Finally, we discovered functional themes related to glycogen metabolism, which may be relevant as a potential mechanism driving the risk of hyperglycemia with statin treatment. Conclusions: The cyno vascular system described here mirrors many of the well-known phenotypes of the human vascular system, and should provide a valuable predictive tool for in vivo studies.

Objective—Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear. Methods and Results—Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow–derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model. Conclusion—These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.