Background:Clinical evidence supports a greater impact of arterial stiffening in cardiovascular mortality in women versus men. Arterial stiffness increases across the menopausal transition, implicating a role of the loss of estrogens in arterial stiffening, but mediating mechanisms remain unclear. Methods:The role of estradiol and smooth muscle cell (SMC) estrogen receptor alpha (ERα) in arterial stiffening, by aortic pulse wave velocity (PWV), was assessed in 3 models: (1) the loss of estradiol in young, female mice comparing sham surgery or bilateral ovariectomy (OVEX) ± estradiol, (2) the impact of sham versus OVEX surgery in young, female SMC-ERα-intact and SMC-ERα-knockout (KO) littermates, and (3) arterial stiffening during natural aging by comparing young and aged, female and male SMC-ERα-intact and SMC-ERα-KO littermates. Mechanistic pathways were assessed using histological assessment of aortic fibrosis and elastin degradation, aortic MMP expression, and atomic force microscopy. Results:OVEX increased PWV and aortic medial fibrosis, with no impact on elastin integrity, in young female mice. Arterial stiffening and fibrosis were prevented in OVEX mice that were supplemented with estradiol. OVEX-induced arterial stiffening in SMC-ERα-intact female mice was prevented in SMC-ERα-KO littermates. In this model, OVEX was also associated with increased aortic medial fibrosis without changes in elastin integrity. Aging from 3 to 18 months significantly increased PWV in female and male SMC-ERα-intact mice. Aging-induced stiffening was fully prevented in female and partially prevented in male SMC-ERα-KO mice. SMC-ERα contributes to aging-associated arterial stiffening by sex-specific mechanisms, including elastin degradation in females and phenotypic changes in SMC stiffness and probability to form cellular adhesions in males. Circulating estradiol was significantly decreased in serum from aged compared with young female mice. Conclusions:These findings support that SMC-ERα contributes to arterial stiffening in female and male mice in situations where the vasculature is exposed to low levels of estradiol.

Endothelial cell mineralocorticoid receptor (EC-MR) is a central regulator of vascular dysfunction beyond its classical renal role. This review summarizes recent advances in our understanding of how EC-MR disrupts vasomotor control via EnNaC activation, eNOS dysregulation and glycocalyx injury, amplifies inflammation and oxidative stress via SGK1, NLRP3 and mitochondrial ROS, and drives fibrosis and remodeling via TGF- β. Through these mechanisms EC-MR contributes to diabetes- and obesity-related vascular disease, acute and chronic kidney injury, and to new disorders including ocular, skin and hypoxic pulmonary diseases, reproductive vascular physiology, cerebrovascular dysfunction, COVID-19 vasculopathy, and cardiac dysfunction. Evidence from preclinical and clinical studies supports both traditional and novel nonsteroidal MR antagonists as potential therapies for these additional indications and suggests endothelial biomarkers to guide translation.

Background/Objectives: Carvedilol is an adrenergic blocker FDA-approved to improve outcomes in heart failure with reduced ejection fraction. Clinical trials examining whether carvedilol may be cardioprotective in the setting of cancer therapy-induced heart failure have generated mixed results that may depend on the cancer regimen, tumor, or comorbidities. Methods: To investigate the therapeutic potential of carvedilol to mitigate doxorubicin cardiotoxicity in cardiomyocytes, myocardial tissue, and in vivo, independent of confounding factors in clinical studies, we utilized disease-free cardiac slices and cardiomyocytes from mice, dogs, and human in vitro, and in wildtype mice injected with doxorubicin in vivo. We further evaluated the impact of carvedilol in dogs with cancer receiving doxorubicin. Results: In primary canine and murine cardiac slices, carvedilol treatment restored autophagy and prevented apoptosis from doxorubicin. Carvedilol restored mitochondrial energetics in human, canine, and murine models. In wildtype mice challenged with doxorubicin, carvedilol prevented declines in cardiac function and alterations in cardiac structure. In pet dogs with cancer and undergoing doxorubicin treatment, carvedilol was beneficial in preserving cardiac function and structure. Conclusions: Carvedilol activates cardioprotective autophagy, arrests doxorubicin-induced cell death, and improves energetics and cardiac structure and function across species.

Hypertension prevalence rises dramatically with advancing age, is not well controlled with current therapy, and contributes substantially to cardiovascular, renal, and neurological disorders that are common in the elderly. The renin-angiotensin-aldosterone system is a hormonal pathway with multiorgan involvement critical to controlling blood pressure. The production of the steroid hormone aldosterone and the activation state of its MR (mineralocorticoid receptor) are important clinical targets for hypertension treatment and cardiorenal disease prevention. This review summarizes studies demonstrating that aging is associated with (1) dysregulation of adrenal aldosterone production by autonomous aldosterone-producing adrenal cells and, when comorbid with obesity, by factors released from adipose tissue that promote adrenal aldosterone production; (2) increased expression of the MR due to oxidative stress-activated and inflammation-activated transcription factors; and (3) aldosterone-independent MR activation by oxidative stress-activated Rac1 (Ras-related C3 botulinum toxin substrate 1), angiotensin II signaling, and declining expression of the cortisol-inactivating enzyme 11β-HSD2 (11-beta-hydroxysteroid dehydrogenase type 2). Together, these data support the concept that elderly individuals are at high risk for mineralocorticoid-driven hypertension and associated cardiovascular, renal, and neurological disease. The review further describes the different classes of agents that inhibit this pathway, including traditional steroidal MR antagonists, newer nonsteroidal MR antagonists, and aldosterone synthase inhibitors, comparing their modes of action. The steroidal MR antagonists and nonsteroidal MR antagonists have different degrees of MR selectivity and potency, yet they all block MR activation by aldosterone, cortisol, and ligand-independent mechanisms. The aldosterone synthase inhibitors block aldosterone production in the adrenal gland and attenuate aldosterone-mediated MR effects. All 3 drug classes raise potassium proportional to the degree of renal MR inhibition. Trials are summarized showing efficacy of the new agents in reducing MR activation, aldosterone production, blood pressure, and adverse cardiorenal outcomes. Head-to-head studies in older individuals are needed to determine the relative efficacy of aldosterone synthase versus MR inhibition for blood pressure control to improve outcomes in the elderly and very old.

BACKGROUND:Atherosclerotic plaque inflammation correlates with risk of rupture, causing myocardial infarction. Lower myocardial infarction risk in young women compared with men abates post-menopause, implicating ERs (estrogen receptors). The ERa (ER alpha) is necessary for estrogen effects on atherosclerosis in mouse models, yet the mechanistic role of ERa in plaque inflammation in both sexes remains unclear. METHODS:The role of ERa in driving endothelial cell (EC) adhesion molecule expression and inflammation was studied in vitro in primary human ECs and in vivo in mice. LDLR (low-density lipoprotein receptor)-knockout mice with EC-specific ERa knockout were compared with ERa-intact littermates after 12 weeks of high-fat diet. RESULTS:In both sexes, EC-specific ERa knockout increased plaque inflammation and expression of adhesion molecules, including ICAM1 (intracellular adhesion molecule 1). In vitro, primary human ECs from young women expressed more ERa and less ICAM1 versus age-matched cells from men. ERa knockdown in human coronary ECs from both sexes increased adhesion molecules. Because the MR (mineralocorticoid receptor) has been implicated in ICAM1 expression and plaque inflammation in males, the impact of ERa on MR-induced ICAM1 expression was explored. In human ECs, estrogen prevented aldosterone induction of ICAM1 and MR enrichment on the ICAM1 promoter. In vivo, EC-specific MR-knockout and EC-ERa/MR-double-knockout/LDLR-knockout mice were studied as above. In females, EC-specific MR knockout did not impact ICAM1 or plaque inflammation, consistent with ERa inhibiting MR function. In the double-knockout model, the lack of MR prevented the increased inflammation and ICAM1 expression observed with loss of EC-ERa. In males, EC-specific MR knockout alone decreased inflammation and ICAM1. In the double-knockout model, the proinflammatory effects of MR and the anti-inflammatory impact of ERa offset each other. CONCLUSIONS:These findings reveal a role for ERa in regulating plaque inflammation in both sexes. In females, estrogen acts via EC-ERa to inhibit MR transcriptional upregulation of ICAM1, attenuating plaque inflammation. In males, ICAM1 expression is driven by the MR and inhibited by ERa.

Cardiovascular disease (CVD) is the leading cause of death in both men and women, but there are sex differences in the timing and mechanisms of disease development. Sex differences particularly influence the development of CVD in the presence of aging and obesity, 2 major risk factors of CVD. The mineralocorticoid and estrogen receptors have been identified as important regulators of vascular function in healthy and disease states. Recent evidence has highlighted interactions between these receptors in the vasculature, and innovations in global and cell-specific knockout mouse models have substantially advanced our understanding of sex-dependent roles of these receptors in vascular health and disease. This review summarizes recent advances in the sex-dependent roles of the mineralocorticoid and estrogen receptors in arterial stiffness and vasomotor dysfunction, 2 early markers of CVD development. These vascular outcomes are examined in the context of aging and obesity, 2 of the most prevalent CVD risk factors. SIGNIFICANCE STATEMENT: Cardiovascular disease (CVD) is the leading cause of death globally for women and men, but there are sex differences in the timing of CVD development across the lifespan and in mechanisms driving disease. This review summarizes sex-specific roles of mineralocorticoid and estrogen receptors in arterial stiffness and vasomotor dysfunction during aging and obesity. Understanding sex-specific mechanisms of CVD is critical to developing precision medicine strategies to prevent and treat CVD in women and men.

BACKGROUND:Imatinib, the first Abl-tyrosine kinase inhibitor (TKI), improved leukemia outcomes without cardiovascular side effects. Newer agents, including ponatinib, addressed imatinib resistance, improving cancer remission, but substantially increased arterial thrombotic events, including myocardial infarction (MI) and stroke. The mechanism behind ponatinib-induced thrombosis and the cardiovascular effect of asciminib, a newly approved Abl-TKI, remain unknown. METHODS:The effect of clinically relevant plasma concentrations of imatinib, ponatinib, and asciminib were compared with vehicle in vivo using SR-BI-mut/LDLR-knockout (KO) mice to assess spontaneous MI and stroke risk. The mechanism was interrogated in C57BL/6J mice, assessing leukocyte trafficking and thromboinflammation by intravital microscopy and flow cytometry, respectively, and in ApoE-KO mice, assessing plaque phenotype by flow cytometry and histology. In vitro effects on human umbilical vein endothelial cells (ECs) and human coronary artery ECs were determined by flow cytometry, PCR, and immunoblotting. The role of TNF (tumor necrosis factor) signaling was evaluated by pharmacological inhibition and small interfering RNA knockdown. RESULTS:In SR-BI-mut/LDLR-KO mice, ponatinib significantly accelerated death from MI and stroke compared with vehicle, imatinib, and asciminib. In human ECs, only ponatinib increased expression of TNF receptors (TNFRs) and adhesion molecules (P-selectin, ICAM1 [intercellular adhesion molecule 1], and VCAM1 [vascular cell adhesion molecule 1]). Ponatinib rapidly induced TNFR2 membrane trafficking and TNF signaling in human umbilical vein ECs. TNFR inhibition or TNFR2 knockdown prevented ponatinib induction of EC adhesion molecules. In vivo, ponatinib increased mesenteric vessel adhesion molecules, leukocyte rolling and adhesion to vessels, leukocyte and platelet activation, and platelet-leukocyte aggregates. In ApoE-KO mice, ponatinib increased plaque necrotic core and inflammation, consistent with a rupture-prone phenotype. Asciminib-treated mice developed none of these in vitro or in vivo toxicities. In C57BL/6J mice, TNFR inhibition blocked ponatinib-induced mesenteric adhesion molecule expression and leukocyte trafficking, but not platelet-leukocyte aggregation. TNFR blockade prevented ponatinib-induced plaque inflammation in ApoE-KO mice and MI and stroke in SR-BI-mut/LDLR-KO mice. CONCLUSIONS:Ponatinib, a potent anticancer therapy, activates ECs, platelets, and leukocytes, driving plaque inflammation and death from MI and stroke in mice, mirroring clinical cardiotoxicities in patients with cancer. Asciminib did not induce these effects, suggesting it might be a safer option for imatinib-resistant patients with cancer. Inhibition of TNFR-mediated endothelial activation is sufficient to prevent ponatinib-induced major adverse cardiovascular events.

BACKGROUND Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer patient survival by inhibiting tumor angiogenesis. However, VEGFRis induce treatment-limiting hypertension which has been associated with impaired vascular endothelial cell (EC) function and kidney damage. The mineralocorticoid receptor (MR) regulates blood pressure (BP) via its effects on the vasculature and the kidney. Thus, we interrogated the role of the MR in EC dysfunction, renal impairment, and hypertension in a mouse model of VEGFRi-induced hypertension using sorafenib.METHODS EC dysfunction in mesenteric arterioles was assessed by immunoblotting for phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1177. Renal damage was measured by assessing glomerular endotheliosis histologically. BP was measured using implanted radiotelemetry.RESULTS Six days of sorafenib treatment significantly impaired mesenteric resistance vessel EC function, induced renal damage, and increased BP. Pharmacologic MR blockade with spironolactone prevented the sorafenib-induced decline in eNOS phosphorylation and renal glomerular endotheliosis, without affecting systolic BP (SBP) or diastolic BP. Mice with the MR knocked out specifically in ECs (EC-MR-KO) were protected from sorafenib-induced EC dysfunction and glomerular endotheliosis, whereas smooth muscle cell-specific MR (SMC-MR) knockout mice were not. Neither EC-MR nor SMC-MR knockout affected the degree to which sorafenib increased SBP or diastolic BP.CONCLUSIONS These results reveal that the MR, specifically in EC but not in SMCs, is necessary for VEGFRi-induced renal and vascular injury. While ineffective at lowering SBP, these data suggest potential therapeutic benefits of MR antagonists, like spironolactone, to protect the vasculature and the kidneys from VEGFRi-induced injury.

Anthracyclines are an effective treatment for hematologic malignancies but have significant risk of cardiotoxicity that increases with lifetime dose, best characterized for doxorubicin. However, patients may receive different anthracycline formulations, and cardiotoxic dose equivalency for nondoxorubicin anthracyclines is uncertain, making it challenging to calculate a patient's lifetime dose to make decisions about safety of additional anthracycline treatment and risk stratification for monitoring and cardioprotective strategies. In addition, dexrazoxane reduces the risk of cardiovascular toxicity with anthracyclines but how to incorporate previous dexrazoxane treatment into risk stratification is unclear. Here, a young woman previously treated with anthracyclines as induction therapy for acute myeloid leukemia and concern for previous anthracycline cardiotoxicity presented with disease relapse. Because optimal therapy included retreatment with anthracyclines, this case demonstrates the multidisciplinary discussion for her case, reviewing the risks and benefits of repeat anthracycline exposure and detailing the evidence for strategies to monitor and prevent worsening cardiotoxicity.

ABSTRACT Background Hypertension is documented in dogs with cancer receiving toceranib, but no studies have evaluated left ventricular (LV) systolic function and biomarkers of endothelial function. Objectives To characterize changes in echocardiographic variables and biomarkers of endothelial function in dogs treated with toceranib. Animals Twenty‐six client‐owned dogs with no evidence of pre‐existing cardiac disease or systemic hypertension are receiving a single agent toceranib for cancer treatment. Methods Dogs were enrolled in this prospective observational study with study visits at baseline, 1, 3, and 5 months after starting toceranib for echocardiographic exams, blood and urine collection, and blood pressure measurements, with an additional blood pressure obtained 2 weeks after starting toceranib. Serum markers of vascular endothelial function (VEGF, endothelin‐1, platelet derived growth factor [PDGF], prostacyclin, cyclic guanosine monophosphate [cGMP]) and urinary nitrate were evaluated with ELISA. Results Dogs were enrolled between 2019 and 2023. Systolic blood pressure increased 2 weeks after initiating toceranib treatment (p = 0.009). Serum prostacyclin concentration was lower after 1 month of treatment (mean 98.8 pg/mL vs. 140.0 pg/mL at baseline, p = 0.03), and serum VEGF concentration was higher after 3 months of treatment (mean of 247.8 pg/mL vs. 135.4 pg/mL at baseline, p = 0.01). Global longitudinal strain (GLS) decreased at the five‐month time point (mean −14.5% vs. −15.7% at baseline, p = 0.048) with no significant change in LV fractional shortening by M‐mode or ejection fraction by Simpson's method of discs. Conclusions Dogs treated with toceranib might have higher systemic blood pressure associated with changes in VEGF and prostacyclin and decreased systolic function.

Inhibitors targeting Abl kinase have dramatically improved survival in Philadelphia chromosome-positive leukemias. First-generation imatinib has minimal cardiovascular side effects, whereas newer agents such as dasatinib, ponatinib, and nilotinib are more effective cancer treatments but carry a high risk of arterial thrombosis. The allosteric Abl kinase inhibitor asciminib was recently approved without long-term cardiovascular follow-up. Previous studies reveal disparate effects of dasatinib, ponatinib, and nilotinib on platelets with consistent evidence of endothelial cell (EC) toxicity. Here, we explore prothrombotic endothelial toxicity mechanisms by comparing exposure to vehicle vs clinically relevant concentrations of imatinib, dasatinib, ponatinib, nilotinib, and asciminib on primary human coronary artery ECs (HCAEC) and mouse models of endothelial injury and vascular thrombosis. Dasatinib and ponatinib increased adhesion of human platelets to HCAEC, specifically when ECs, but not platelets, were exposed to drugs. Dasatinib, ponatinib, and nilotinib impaired HCAEC healing in vitro, whereas only nilotinib impaired healing in vivo and increased von Willebrand factor levels in mice. Dasatinib and ponatinib increased early platelet but not fibrin accumulation in the mouse cremaster arteriole laser injury-induced thrombosis model, and only ponatinib increased platelet-leukocyte aggregate formation. Asciminib had no toxic effect in any of these assays, similar to imatinib. These studies reveal novel and distinct mechanisms by which EC damage induced by dasatinib, ponatinib, and nilotinib contributes to a prothrombogenic EC state. The findings suggest the need for distinct side effect prevention strategies and provide preclinical data supporting vascular safety of asciminib, while awaiting long-term vascular safety follow-up results.

Peripheral artery disease (PAD) is the narrowing of the arteries that carry blood to the lower extremities. PAD has been traditionally associated with atherosclerosis. However, recent studies have found that thrombotic events triggered by medial arterial calcification (MAC) is the primary cause of chronic limb ischemia below the knee. MAC is localized around the elastic fibers surrounding smooth muscle cells (SMCs) in arteries. Matrix GLA protein (MGP) binds circulating calcium and prevents hydroxyapatite mineral deposition, while also modulating pro-osteogenic signaling by attenuating bone morphogenetic protein (BMP)-2-mediated activation of Runx2 gene expression. Mgp-/- mice develop severe MAC and die around 8 wk after birth due to aortic rupture or heart failure. We previously discovered a rare genetic disease, arterial calcification due to deficiency of CD73 (ACDC), in which patients present with extensive MAC in their lower extremity arteries. Using a patient-specific induced pluripotent stem cell model, we found that rapamycin (RAPA) inhibited calcification. Here, we investigated whether rapamycin could reduce MAC in vivo using the Mgp-/- murine model. Mgp+/+ and Mgp-/- mice received 5 mg/kg rapamycin or vehicle. Calcification content was assessed via microCT, and vascular morphology and extracellular matrix content were assessed histologically. Immunostaining and Western blot analysis were used to examine SMC phenotype and extracellular matrix content. Rapamycin prolonged Mgp-/- mice lifespan, decreased mineral density in the arteries, maintained SMC contractile phenotype, and improved vessel structure, however, calcification volume was unchanged. Mgp-/- mice with SMC-specific deletion of Raptor or Rictor did not recapitulate treatment with rapamycin. These findings suggest rapamycin promotes beneficial vascular remodeling in vessels with MAC.NEW & NOTEWORTHY Peripheral artery disease (PAD) is associated with medial arterial calcification (MAC), which involves calcification of arterial elastic fibers and smooth muscle cells (SMCs). Matrix GLA protein (MGP) inhibits vascular calcification, and Mgp-/- mice develop severe MAC. Using this model, we found rapamycin (RAPA) prolonged lifespan, reduced arterial mineral density, maintained SMC contractile phenotype, and improved vessel structure, though calcification volume remained unchanged. Findings highlight rapamycin's potential for vascular remodeling in MAC.

Arterial stiffness is associated with overall and cardiovascular-specific mortality, and this association is exacerbated in women over 55 yr of age. Recent literature supports that stimulation of the angiotensin II type 2 receptor (AT2R) can protect from arterial stiffening, and that AT2R has a greater role in female cardiovascular physiology relative to males. The current study aimed to investigate the role of the AT2R in sex differences in aging-associated arterial stiffness. In female mice, the aging-related increase in arterial stiffness is temporally associated with a loss of aortic AT2R mRNA expression, but this is not observed in males. Chronic AT2R inhibition in vivo increases arterial stiffening in young female and male mice, as well as middle-aged female mice. The inhibition of AT2R is associated with an increase in aortic integrinα5 mRNA expression in young males and an increase in collagen1α1 mRNA expression in middle-aged females. Overall, these findings identify a sex-specific mechanism of aging-associated arterial stiffening in mice involving AT2R attenuation and collagen upregulation in females.NEW & NOTEWORTHY This is the first investigation to show that the angiotensin II type 2 receptor (AT2R) attenuates aging-associated arterial stiffness in middle-aged female mice. The AT2R also attenuates arterial stiffness in young female and male mice, but there are sex-specific molecular mechanisms that contribute to the role of AT2R in vessel stiffening.

HomeCirculationVol. 149, No. 13Aggregation and Contextualization of Murine Investigations Improves Discovery of Significant Human Atherosclerotic Cardiovascular Disease Associations No AccessLetterRequest AccessFull TextAboutView Full TextView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toNo AccessLetterRequest AccessFull TextAggregation and Contextualization of Murine Investigations Improves Discovery of Significant Human Atherosclerotic Cardiovascular Disease Associations Megan M. Shuey, Yihua Wang, Rachel R. Xiang, Aaron Zou, Protiva Rahman, Daniel Fabbri, Joshua A. Beckman, Iris Z. Jaffe and Quinn S. Wells Megan M. ShueyMegan M. Shuey https://orcid.org/0000-0003-2866-3562 Division of Genetic Medicine, Vanderbilt University Medical Center, Nashville, TN (M.M.S.). , Yihua WangYihua Wang Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA (Y.W., R.R.X., A.Z., I.Z.J.). , Rachel R. XiangRachel R. Xiang https://orcid.org/0000-0002-2316-9293 Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA (Y.W., R.R.X., A.Z., I.Z.J.). , Aaron ZouAaron Zou Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA (Y.W., R.R.X., A.Z., I.Z.J.). , Protiva RahmanProtiva Rahman Department of Biomedical Informatics (P.R., D.F., Q.S.W.), Vanderbilt University Medical Center, Nashville, TN. , Daniel FabbriDaniel Fabbri https://orcid.org/0000-0003-0530-2510 Department of Biomedical Informatics (P.R., D.F., Q.S.W.), Vanderbilt University Medical Center, Nashville, TN. , Joshua A. BeckmanJoshua A. Beckman https://orcid.org/0000-0001-8332-8439 Division of Cardiovascular Medicine (J.A.B., Q.S.W.), Vanderbilt University Medical Center, Nashville, TN. , Iris Z. JaffeIris Z. Jaffe https://orcid.org/0000-0001-9300-1253 Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA (Y.W., R.R.X., A.Z., I.Z.J.). and Quinn S. WellsQuinn S. Wells Correspondence to: Quinn S. Wells, MD, PharmD, MSCI, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 2525 West End Ave, Ste 300, Nashville, TN 37203. Email E-mail Address: [email protected] Department of Biomedical Informatics (P.R., D.F., Q.S.W.), Vanderbilt University Medical Center, Nashville, TN. Division of Cardiovascular Medicine (J.A.B., Q.S.W.), Vanderbilt University Medical Center, Nashville, TN. Originally published25 Mar 2024https://doi.org/10.1161/CIRCULATIONAHA.123.067510Circulation. 2024;149:1056–1058Footnotes*M.M. Shuey and Y. Wang contributed equally.†J.A. Beckman, I.Z. Jaffe, and Q.S. Wells contributed equally.This manuscript was sent to Andrew Baker, Guest Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page 1058.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Quinn S. Wells, MD, PharmD, MSCI, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 2525 West End Ave, Ste 300, Nashville, TN 37203. Email quinn.s.wells@vumc.orgREFERENCES1. Daugherty A, Tall AR, Daemen M, Falk E, Fisher EA, Garcia-Cardena G, Lusis AJ, Owens AP, Rosenfeld ME, Virmani R, et al. Recommendation on design, execution, and reporting of animal atherosclerosis studies: a scientific statement from the American Heart Association.Arterioscler Thromb Vasc Biol. 2017; 37:e131–e157. doi: 10.1161/ATV.0000000000000062LinkGoogle Scholar2. von Scheidt M, Zhao Y, Kurt Z, Pan C, Zeng L, Yang X, Schunkert H, Lusis AJ. Applications and limitations of mouse models for understanding human atherosclerosis.Cell Metab. 2017; 25:248–261. doi: 10.1016/j.cmet.2016.11.001CrossrefMedlineGoogle Scholar3. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis.Nature. 2011; 473:317–325. doi: 10.1038/nature10146CrossrefMedlineGoogle Scholar4. Shuey MM, Xiang RR, Moss ME, Carvajal BV, Wang Y, Camarda N, Fabbri D, Rahman P, Ramsey J, Stepanian A, et al. Systems approach to integrating preclinical apolipoprotein E-knockout investigations reveals novel etiologic pathways and master atherosclerosis network in humans.Arterioscler Thromb Vasc Biol. 2022; 42:35–48. doi: 10.1161/ATVBAHA.121.317071LinkGoogle Scholar5. Xiang RR, Wang Y, Shuey MM, Brigett Carvajal BV, Wells QS, Beckman JA, Jaffe IZ. Development and implementation of an integrated preclinical atherosclerosis database.Circ Genom Precis Med. 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Carvedilol (CAR) is an FDA-approved adrenergic blocker commonly given to cardiovascular disease patients. Interestingly, CAR also shows anti-cancer potential in reducing cancer-specific mortality in breast cancer patients. To advance CAR usage in cardio-oncology, we hypothesize that elucidating the molecular mechanism of the energy sensing and homeostasis pathways underlying CAR’s cardioprotective capabilities can enhance its clinical translation. Canine cardiac slices were generated from 3 euthanized pet dogs free of cardiovascular disease. CAR pretreatment (1μM, 4 hrs) with or without a 24-hr exposure to 5μM of cardiotoxic doxorubicin (DOX) was performed. Murine slices from 5 C57Bl6 mice with the same treatments as above was also performed. Direct tissue imaging on IVIS Spectrum was carried out to assess apoptosis by Annexin V staining, autophagy with autophagy detecting nanoparticle (ADN) developed in-house, and DOX retention in tissue by the inherent DOX fluorescence. Tissue was analyzed by histological staining of apoptosis and Western blot of autophagy and energy sensing pathways. Energetics quantified by Seahorse assay was performed in isolated mitochondria from cardiac slices in rat cardiomyoblasts (H9C2 cells) and human iPSC-induced cardiomyocytes (iCells). In both canine and murine cardiac slices, autophagy was significantly reduced (p<0.05) by DOX while apoptosis was significantly increased (p<0.05), both of which were reversed by CAR. CAR did not change DOX fluorescence in tissue. Western blot of autophagy biomarkers LC3, p62 and Beclin-1 confirmed that autophagy impaired by DOX was restored by CAR. The beneficial autophagy restoration by CAR was modulated by a significantly increased phosphorylation of AMP kinase (AMPK), a key energy sensing pathway that activates autophagy via significantly reducing mTOR (p<0.0001). In mitochondria isolated from cardiac slices, basal respiration and ATP production that were significantly impaired by DOX were rescued (p<0.0001) by CAR. Similar rescue of basal respiration and ATP production by CAR was seen in murine and human cardiomyocytes, suggesting that CAR exerts a direct protective effect on the at-risk cardiomyocytes during DOX stress. We demonstrated for the first time in human, canine, and murine that CAR targets the AMPK pathway to activate autophagy, reduce cardiomyocyte apoptosis, and restore mitochondrial energetics, thus with immense translational potential in cardioprotection.

Background: Atherosclerosis (athero) inflammation predicts plaque rupture, causing heart attack and stroke. Female protection from heart attack and stroke is lost with age, implicating estrogen, but by unclear mechanism. Our lab showed that the mineralocorticoid receptor (MR) is expressed in endothelial cells (ECs), where it drives plaque inflammation in male mice by inducing the NFkB-regulated adhesion molecule ICAM1. Females had less inflamed plaques, unchanged by EC-MR-knockout (KO). Hypothesis: We hypothesized that EC-estrogen receptor alpha (ER) protects females from plaque inflammation by inhibiting NFkB and EC-MR induction of ICAM1. Methods and Results: Mice with EC specific MR KO (EC-MR-KO), ER KO (EC-ER-KO), or EC KO of both receptors (DKO) were developed and crossed to the LDLR KO background. EC specific gene recombination of MR and/or ER was confirmed by genomic PCR of lung (rich in ECs) and compared to blood, revealing no recombination in leukocytes. Baseline athero risk factors were unchanged by genotype. Male and female mice of all KO lines and floxed Cre- littermate controls were fed high fat diet for 12 weeks resulting in increased body weight and cholesterol with no difference by genotype in athero risk factors. Aortic arches were digested for flow cytometry to quantify plaque inflammation, confirming less inflamed plaques in females versus males. EC-ER-KO increased plaque inflammation, while DKO prevented this increase in both sexes. Compared to controls, ICAM1 protein expression in the descending aorta was decreased by EC-MR-KO only in males. In both sexes, ICAM1 was increased by EC-ER-KO and unchanged by DKO. Females had increased aortic expression of IkB, the inhibitor of NFkB, compared to males and this was prevented by EC-ER-KO. In primary human aortic ECs (HAECs) in vitro , cells from females expressed significantly less ICAM1 protein compared to males. Conclusions: Female atherogenic mice have less inflamed plaques than males and female HAECs express less ICAM1. In vivo , EC-ER protects from plaque inflammation and suppresses ICAM1 in both sexes and IkB in females, and EC-MR is necessary for this atheroprotection. These data support the concept that EC-ER prevents inflammation by inhibiting NFkB and EC-MR-induced ICAM1 expression.

Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer survival but are associated with treatment-limiting hypertension, often attributed to endothelial cell (EC) dysfunction. Using phosphoproteomic profiling of VEGFRi-treated ECs, drugs were screened for mitigators of VEGFRi-induced EC dysfunction and validated in primary aortic ECs, mice, and canine cancer patients. VEGFRi treatment significantly raised systolic blood pressure (SBP) and increased markers of endothelial and renal dysfunction in mice and canine cancer patients. α-Adrenergic-antagonists were identified as drugs that most oppose the VEGFRi proteomic signature. Doxazosin, one such α-antagonist, prevented EC dysfunction in murine, canine, and human aortic ECs. In mice with sorafenib-induced-hypertension, doxazosin mitigated EC dysfunction but not hypertension or glomerular endotheliosis, while lisinopril mitigated hypertension and glomerular endotheliosis without impacting EC function. Hence, reversing EC dysfunction was insufficient to mitigate VEGFRi-induced-hypertension in this mouse model. Canine cancer patients with VEGFRi-induced-hypertension were randomized to doxazosin or lisinopril and both agents significantly decreased SBP. The canine clinical trial supports safety and efficacy of doxazosin and lisinopril as antihypertensives for VEGFRi-induced-hypertension and the potential of trials in canines with spontaneous cancer to accelerate translation. The overall findings demonstrate the utility of phosphoproteomics to identify EC-protective agents to mitigate cardio-oncology side effects.

Vascular stiffness increases with aging, obesity and hypertension and predicts cardiovascular risk. The levels of histone H3-lysine-27 methylation (H3K27me) and the histone methyltransferase EZH2 both decrease in aging vessels, driving vascular stiffness. The impact of EZH2 inhibitors on vascular stiffness is unknown. We tested the hypothesis that the EZH2 inhibitor GSK126, currently in development for cancer treatment, increases vascular stiffness and explored underlying molecular mechanisms. Young (3 month) and middle-aged (12 month) male mice were treated with GSK126 for 1-2 months and primary human aortic smooth muscle cells (HASMCs) from young male and female donors were treated with GSK126 for 24-48 h. Stiffness was measured in vivo by pulse wave velocity and in vitro by atomic force microscopy (AFM) and vascular structure was quantified histologically. Extracellular matrix proteins were studied by qRT-PCR, immunoblotting, zymography and chromatin immunoprecipitation. GSK126 treatment decreased H3K27 methylation (H3K27me) and increased acetylation (H3K27ac) in mouse vessels and in HASMCs. In GSK126-treated mice, aortic stiffness increased without changes in vascular fibrosis. EZH2 inhibition enhanced elastin fiber degradation and matrix metalloprotease-2 (MMP2) expression. In HASMCs, GSK126 treatment increased synthetic phenotype markers and intrinsic HASMCs stiffness by AFM with altered cytoskeletal structure and increased nuclear actin staining. GSK126 also increased MMP2 protein expression, activity and enrichment of H3K27ac at the MMP2 promoter in HASMCs. GSK126 causes vascular stiffening, inducing MMP2 activity, elastin degradation, and modulation of SMC phenotype and cytoskeletal stiffness. These findings suggest that EZH2 inhibitors used to treat cancer could negatively impact the vasculature by enhancing stiffness and merits examination in human trials.

BACKGROUND: Women with a history of preeclampsia have evidence of premature atherosclerosis and increased risk of myocardial infarction and stroke compared with women who had a normotensive pregnancy. Whether this is due to common risk factors or a direct impact of prior preeclampsia exposure has never been tested in a mouse atherosclerosis model. METHODS: Pregnant LDLR-KO (low-density lipoprotein receptor knockout; n=35) female mice were randomized in midgestation to sFlt1 (soluble fms-like tyrosine kinase 1)-expressing adenovirus or identical control adenovirus. Postpartum, mice were fed high-fat diet for 8 weeks to induce atherogenesis. Comparison between the control and preeclampsia models was made for metabolic parameters, atherosclerosis burden and composition by histology, plaque inflammation by flow cytometry, and aortic cytokines and inflammatory markers using a cytokine array. RESULTS: In pregnant LDLR-KO mice, sFlt1 adenovirus significantly induced serum sFlt1, blood pressure, renal endotheliosis, and decreased pup viability. After 8 weeks of postpartum high fat feeding, body weight, fasting glucose, plasma cholesterol, HDL (high-density lipoprotein), and LDL (low-density lipoprotein) were not significantly different between groups with no change in aortic root plaque size, lipid content, or necrotic core area. Flow cytometry demonstrated significantly increased CD45+ aortic arch leukocytes and CD3+T cells and aortic lysate contained more CCL (CC motif chemokine ligand) 22 and fetuin A and decreased expression of IGFBP6 (insulin-like growth factor-binding protein 6) and CCL21 in preeclampsia-exposed mice compared with controls. CONCLUSIONS: In atherogenic LDLR-KO mice, exposure to sFlt1-induced preeclampsia during pregnancy increases future atherosclerotic plaque inflammation, supporting the concept that preeclampsia directly exacerbates atherosclerotic inflammation independent of preexisting risk factors. This mechanism may contribute to ischemic vascular disease in women after preeclampsia pregnancy.