Smooth muscle cell CYB5R3 preserves cardiac and vascular function under chronic hypoxic stress.

Chronic hypoxia is a major driver of cardiovascular complications, including heart failure. The nitric oxide (NO) - soluble guanylyl cyclase (sGC) - cyclic guanosine monophosphate (cGMP) pathway is integral to vascular tone maintenance. Specifically, NO binds its receptor sGC within vascular smooth muscle cells (SMC) in its reduced heme (Fe2+) form to increase intracellular cGMP production, activate protein kinase G (PKG) signaling, and induce vessel relaxation. Under chronic hypoxia, oxidative stress drives oxidation of sGC heme (Fe2+→Fe3+), rendering it NO-insensitive. We previously showed that cytochrome b5 reductase 3 (CYB5R3) in SMC is a sGC reductase important for maintaining NO-dependent vasodilation and conferring resilience to systemic hypertension and sickle cell disease-associated pulmonary hypertension. To test whether CYB5R3 may be protective in the context of chronic hypoxia, we subjected SMC-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 3 weeks hypoxia and assessed vascular and cardiac function using echocardiography, pressure volume loops and wire myography. Hypoxic stress caused 1) biventricular hypertrophy in both WT and SMC CYB5R3 KO, but to a larger degree in KO mice, 2) blunted vasodilation to NO-dependent activation of sGC in coronary and pulmonary arteries of KO mice, and 3) decreased, albeit still normal, cardiac function in KO mice. Overall, these data indicate that SMC CYB5R3 deficiency potentiates bilateral ventricular hypertrophy and blunts NO-dependent vasodilation under chronic hypoxia conditions. This implicates that SMC CYB5R3 KO mice post 3-week hypoxia have early stages of cardiac remodeling and functional changes that could foretell significantly impaired cardiac function with longer exposure to hypoxia.

Antagonism of Forkhead Box Subclass O Transcription Factors Elicits Loss of Soluble Guanylyl Cyclase Expression.

Nitric oxide (NO) stimulates soluble guanylyl cyclase (sGC) activity, leading to elevated intracellular cyclic guanosine 3',5'-monophosphate (cGMP) and subsequent vascular smooth muscle relaxation. It is known that downregulation of sGC expression attenuates vascular dilation and contributes to the pathogenesis of cardiovascular disease. However, it is not well understood how sGC transcription is regulated. Here, we demonstrate that pharmacological inhibition of Forkhead box subclass O (FoxO) transcription factors using the small-molecule inhibitor AS1842856 significantly blunts sGC α and ß mRNA expression by more than 90%. These effects are concentration-dependent and concomitant with greater than 90% reduced expression of the known FoxO transcriptional targets, glucose-6-phosphatase and growth arrest and DNA damage protein 45 α (Gadd45α). Similarly, sGC α and sGC ß protein expression showed a concentration-dependent downregulation. Consistent with the loss of sGC α and ß mRNA and protein expression, pretreatment of vascular smooth muscle cells with the FoxO inhibitor decreased sGC activity measured by cGMP production following stimulation with an NO donor. To determine if FoxO inhibition resulted in a functional impairment in vascular relaxation, we cultured mouse thoracic aortas with the FoxO inhibitor and conducted ex vivo two-pin myography studies. Results showed that aortas have significantly blunted sodium nitroprusside-induced (NO-dependent) vasorelaxation and a 42% decrease in sGC expression after 48-hour FoxO inhibitor treatment. Taken together, these data are the first to identify that FoxO transcription factor activity is necessary for sGC expression and NO-dependent relaxation.

Redox control of vascular smooth muscle cell function and plasticity.

Vascular smooth muscle cells (SMC) play a major role in vascular diseases, such as atherosclerosis and hypertension. It has long been established in vitro that contractile SMC can phenotypically switch to function as proliferative and/or migratory cells in response to stimulation by oxidative stress, growth factors, and inflammatory cytokines. Reactive oxygen species (ROS) are oxidative stressors implicated in driving vascular diseases, shifting cell bioenergetics, and increasing SMC proliferation, migration, and apoptosis. In this review, we summarize our current knowledge of how disruptions to redox balance can functionally change SMC and how this may influence vascular disease pathogenesis. Specifically, we focus on our current understanding of the role of vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) 1, 4, and 5 in SMC function. We also review the evidence implicating mitochondrial fission in SMC phenotypic transitions and mitochondrial fusion in maintenance of SMC homeostasis. Finally, we discuss the importance of the redox regulation of the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway as a potential oxidative and therapeutic target for regulating SMC function.

Redox regulation of soluble guanylyl cyclase.

The nitric oxide/soluble guanylyl cyclase (NO-sGC) signaling pathway regulates the cardiovascular, neuronal, and gastrointestinal systems. Impaired sGC signaling can result in disease and system-wide organ failure. This review seeks to examine the redox control of sGC through heme and cysteine regulation while discussing therapeutic drugs that target various conditions. Heme regulation involves mechanisms of insertion of the heme moiety into the sGC protein, the molecules and proteins that control switching between the oxidized (Fe3+) and reduced states (Fe2+), and the activity of heme degradation. Modifications to cysteine residues by S-nitrosation on the α1 and ß1 subunits of sGC have been shown to be important in sGC signaling. Moreover, redox balance and localization of sGC is thought to control downstream effects. In response to altered sGC activity due to changes in the redox state, many therapeutic drugs have been developed to target decreased NO-sGC signaling. The importance and relevance of sGC continues to grow as sGC dysregulation leads to numerous disease conditions.

Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions.

Atherosclerotic plaque rupture with subsequent embolic events is a major cause of sudden death from myocardial infarction or stroke. Although smooth muscle cells (SMCs) produce and respond to collagens in vitro, there is no direct evidence in vivo that SMCs are a crucial source of collagens and that this impacts lesion development or fibrous cap formation. We sought to determine how conditional SMC-specific knockout of collagen type XV (COL15A1) in SMC lineage tracing mice affects advanced lesion formation given that 1) we have previously identified a Col15a1 sequence variant associated with age-related atherosclerosis, 2) COL15A1 is a matrix organizer enhancing tissue structural integrity, and 3) small interfering RNA-mediated Col15a1 knockdown increased migration and decreased proliferation of cultured human SMCs. We hypothesized that SMC-derived COL15A1 is critical in advanced lesions, specifically in fibrous cap formation. Surprisingly, we demonstrated that SMC-specific Col15a1 knockout mice fed a Western diet for 18 wk failed to form advanced lesions. SMC-specific Col15a1 knockout resulted in lesions reduced in size by 78%, with marked reductions in numbers and proliferating SMCs, and lacked a SMC and extracellular matrix-rich lesion or fibrous cap. In vivo RNA-seq analyses on SMC Col15a1 knockout and wild-type lesions suggested that a mechanism for these effects is through global repression of multiple proatherogenic inflammatory pathways involved in lesion development. These results provide the first direct evidence that a SMC-derived collagen, COL15A1, is critical during lesion pathogenesis, but, contrary to expectations, its loss resulted in marked attenuation rather than exacerbation of lesion pathogenesis.NEW & NOTEWORTHY We report the first direct in vivo evidence that a smooth muscle cell (SMC)-produced collagen, collagen type XV (COL15A1), is critical for atherosclerotic lesion development. SMC Col15a1 knockout markedly attenuated advanced lesion formation, likely through reducing SMC proliferation and impairing multiple proatherogenic inflammatory processes.

Angiotensin II augments renal vascular smooth muscle soluble GC expression via an AT1 receptor-forkhead box subclass O transcription factor signalling axis.

BACKGROUND AND PURPOSE: Reduced renal blood flow triggers activation of the renin-angiotensin-aldosterone system (RAAS) leading to renovascular hypertension. Renal vascular smooth muscle expression of the NO receptor, soluble GC (sGC), modulates the vasodilator response needed to control renal vascular tone and blood flow. Here, we tested if angiotensin II (Ang II) affects sGC expression via an AT1 receptor-forkhead box subclass O (FoxO) transcription factor dependent mechanism. EXPERIMENTAL APPROACH: Using a murine two-kidney-one-clip (2K1C) renovascular hypertension model, we measured renal artery vasodilatory function and sGC expression. Additionally, we conducted cell culture studies using rat renal pre-glomerular smooth muscle cells (RPGSMCs) to test the in vitro mechanistic effects of Ang II treatment on sGC expression and downstream function. KEY RESULTS: Contralateral, unclipped renal arteries in 2K1C mice showed increased NO-dependent vasorelaxation compared to sham control mice. Immunofluorescence studies revealed increased sGC protein expression in 2K1C contralateral renal arteries over sham controls. RPGSMCs treated with Ang II caused a significant up-regulation of sGC mRNA and protein expression as well as downstream sGC-dependent signalling. Ang II signalling effects on sGC expression occurred through an AT1 receptor and FoxO transcription factor-dependent mechanism at both the mRNA and protein expression levels. CONCLUSION AND IMPLICATIONS: Renal artery smooth muscle, in vivo and in vitro, up-regulates expression of sGC following RAAS activity. In both cases, up-regulation of sGC leads to increased downstream cGMP signalling, suggesting a previously unrecognized protective mechanism to improve renal blood flow in the uninjured contralateral renal artery. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Low Rates of Chest Wall Toxicity When Individualizing the Planning Target Volume Margin in Patients With Early Stage Lung Cancer Treated With Stereotactic Body Radiation Therapy.

PURPOSE: Chest wall (CW) toxicity is a potentially debilitating complication of stereotactic body radiation therapy for non-small cell lung cancer, occurring in 10% to 40% of patients. Smaller tumor-to-CW distance has been identified as a risk factor for CW toxicity. We report our experience with individualizing the planning target volume (PTV) along the CW in an effort to reduce the volume of this organ at risk receiving 30 Gy to 50 Gy. METHODS AND MATERIALS: We performed an institutional review board-approved retrospective analysis of patients with stage I (T1-2aN0M0) non-small cell lung cancer who received stereotactic body radiation therapy between June 2009 and July 2016. Four-dimensional computed tomography was used for treatment planning. A uniform 5-mm expansion of the internal target volume was generated for the PTV. Areas of overlap with the CW were removed from the PTV. Treatment was delivered with cone beam computed tomography guidance. CW toxicity was assessed per the Common Terminology Criteria for Adverse Events, version 5. Descriptive statistics were used to analyze outcomes. RESULTS: The median follow-up time was 36.8 months. A total of 260 tumors were treated in 225 patients. 225 tumors in 203 patients were peripheral. The internal target volumes for 143 tumors (63.6%) were located within 5 mm of the CW. The median total dose was 48 Gy (range, 42-60 Gy) in 4 fractions (range, 3-5 fractions). The overall rate of grade 1 to 2 CW toxicity was 2.2%, and 2.8% for tumors located within 5 mm of the CW. There were no grade 3/4 cases and no increase in local recurrences with the use of a truncated PTV with a 3-year local control of 92.1% (95% confidence interval, 87.4%-96.8%). CONCLUSIONS: Truncation of the PTV margin along the CW resulted in a marked reduction of CW toxicity for tumors in close proximity to the CW, with only a 2.8% rate of grade 1 to 2 CW toxicity. Despite PTV reduction, there was no appreciable increase in local failures. A multi-institutional validation of this technique is needed before general incorporation into clinical practice.

Assessment of a Contralateral Esophagus-Sparing Technique in Locally Advanced Lung Cancer Treated With High-Dose Chemoradiation: A Phase 1 Nonrandomized Clinical Trial.

IMPORTANCE: Severe acute esophagitis occurs in up to 20% of patients with locally advanced lung cancer treated with chemoradiation therapy to at least 60 Gy once daily and represents a dose-limiting toxic event associated with poor outcomes. OBJECTIVE: To assess whether formalized sparing of the contralateral esophagus (CE) is associated with reduced risk of severe acute esophagitis. DESIGN, SETTING, AND PARTICIPANTS: This single-center phase 1 nonrandomized clinical trial assessing an empirical CE-sparing technique enrolled patients from July 2015 to January 2019. In total, 27 patients with locally advanced non-small cell lung carcinoma (with or without solitary brain metastasis) or limited-stage small cell lung carcinoma with gross tumor within 1 cm of the esophagus were eligible. INTERVENTIONS: Intensity-modulated radiation therapy to 70 Gy at 2 Gy/fraction concurrent with standard chemotherapy with or without adjuvant durvalumab. The esophageal wall contralateral to gross tumor was contoured as an avoidance structure to guide a steep dose falloff gradient. Target coverage was prioritized over CE sparing, and 99% of internal and planning target volumes had to be covered by 70 Gy and at least 63 Gy, respectively. MAIN OUTCOMES AND MEASURES: The primary end point was the rate of at least grade 3 acute esophagitis as assessed by Common Terminology Criteria for Adverse Events, version 4. RESULTS: Of 27 patients enrolled, 25 completed chemoradiation therapy. Nineteen patients had non-small cell lung carcinoma, and 6 had small cell lung carcinoma. The median age at diagnosis was 67 years (range, 51-81 years), and 15 patients (60%) were men. Thirteen patients (52%) had stage IIIA cancer, 10 (40%) had stage IIIB cancer, and 2 (8%) had stage IV cancer. The median CE maximum dose was 66 Gy (range, 44-71 Gy); the median volume of CE receiving at least 55 Gy was 1.4 cm3 (range, 0-5.3 cm3), and the median volume of CE receiving at least 45 Gy was 2.7 cm3 (range, 0-9.2 cm3). The median combined percentage of lung receiving at least 20 Gy was 25% (range, 11%-37%). The median follow-up was 33.3 months (range, 11.1-52.2 months). Among the 20 patients who had treatment breaks of 0 to 3 days and were thus evaluable for the primary end point, the rate of at least grade 3 esophagitis was 0%. Other toxic events observed among all 25 patients included 7 (28%) with grade 2 esophagitis, 3 (12%) with at least grade 2 pneumonitis (including 1 with grade 5), and 2 (8%) with at least grade 3 cardiac toxic event (including 1 with grade 5). There was no isolated local tumor failure. The 2-year progression-free survival rate was 57% (95% CI, 33%-75%), and the 2-year overall survival rate was 67% (95% CI, 45%-82%). CONCLUSIONS AND RELEVANCE: This phase 1 nonrandomized clinical trial found that the CE-sparing technique was associated with reduced risk of esophagitis among patients treated uniformly with chemoradiation therapy (to 70 Gy), with no grade 3 or higher esophagitis despite tumor within 1 cm of the esophagus. This technique may be translated into clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02394548.

Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.

Nitric oxide regulates BP by binding the reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) and relaxing vascular smooth muscle cells (SMCs). We previously showed that sGC heme iron reduction (Fe3+ → Fe2+) is modulated by cytochrome b5 reductase 3 (CYB5R3). However, the in vivo role of SMC CYB5R3 in BP regulation remains elusive. Here, we generated conditional smooth muscle cell-specific Cyb5r3 KO mice (SMC CYB5R3-KO) to test if SMC CYB5R3 loss affects systemic BP in normotension and hypertension via regulation of the sGC redox state. SMC CYB5R3-KO mice exhibited a 5.84-mmHg increase in BP and impaired acetylcholine-induced vasodilation in mesenteric arteries compared with controls. To drive sGC oxidation and elevate BP, we infused mice with angiotensin II. We found that SMC CYB5R3-KO mice exhibited a 14.75-mmHg BP increase, and mesenteric arteries had diminished nitric oxide-dependent vasodilation but increased responsiveness to sGC heme-independent activator BAY 58-2667 over controls. Furthermore, acute injection of BAY 58-2667 in angiotensin II-treated SMC CYB5R3-KO mice showed greater BP reduction compared with controls. Together, these data provide the first in vivo evidence to our knowledge that SMC CYB5R3 is an sGC heme reductase in resistance arteries and provides resilience against systemic hypertension development.

Smooth muscle cytochrome b5 reductase 3 deficiency accelerates pulmonary hypertension development in sickle cell mice.

Pulmonary and systemic vasculopathies are significant risk factors for early morbidity and death in patients with sickle cell disease (SCD). An underlying mechanism of SCD vasculopathy is vascular smooth muscle (VSM) nitric oxide (NO) resistance, which is mediated by NO scavenging reactions with plasma hemoglobin (Hb) and reactive oxygen species that can oxidize soluble guanylyl cyclase (sGC), the NO receptor. Prior studies show that cytochrome b5 reductase 3 (CYB5R3), known as methemoglobin reductase in erythrocytes, functions in VSM as an sGC heme iron reductase critical for reducing and sensitizing sGC to NO and generating cyclic guanosine monophosphate for vasodilation. Therefore, we hypothesized that VSM CYB5R3 deficiency accelerates development of pulmonary hypertension (PH) in SCD. Bone marrow transplant was used to create SCD chimeric mice with background smooth muscle cell (SMC)-specific tamoxifen-inducible Cyb5r3 knockout (SMC R3 KO) and wild-type (WT) control. Three weeks after completing tamoxifen treatment, we observed 60% knockdown of pulmonary arterial SMC CYB5R3, 5 to 6 mm Hg elevated right-ventricular (RV) maximum systolic pressure (RVmaxSP) and biventricular hypertrophy in SS chimeras with SMC R3 KO (SS/R3KD) relative to WT (SS/R3WT). RV contractility, heart rate, hematological parameters, and cell-free Hb were similar between groups. When identically generated SS/R3 chimeras were studied 12 weeks after completing tamoxifen treatment, RVmaxSP in SS/R3KD had not increased further, but RV hypertrophy relative to SS/R3WT persisted. These are the first studies to establish involvement of SMC CYB5R3 in SCD-associated development of PH, which can exist in mice by 5 weeks of SMC CYB5R3 protein deficiency.

Interleukin-1ß has atheroprotective effects in advanced atherosclerotic lesions of mice.

Despite decades of research, our understanding of the processes controlling late-stage atherosclerotic plaque stability remains poor. A prevailing hypothesis is that reducing inflammation may improve advanced plaque stability, as recently tested in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial, in which post-myocardial infarction subjects were treated with an IL-1ß antibody. Here, we performed intervention studies in which smooth muscle cell (SMC) lineage-tracing Apoe-/- mice with advanced atherosclerosis were treated with anti-IL-1ß or IgG control antibodies. Surprisingly, we found that IL-1ß antibody treatment between 18 and 26 weeks of Western diet feeding induced a marked reduction in SMC and collagen content, but increased macrophage numbers in the fibrous cap. Moreover, although IL-1ß antibody treatment had no effect on lesion size, it completely inhibited beneficial outward remodeling. We also found that SMC-specific knockout of Il1r1 (encoding IL-1 receptor type 1) resulted in smaller lesions nearly devoid of SMCs and lacking a fibrous cap, whereas macrophage-selective loss of IL-1R1 had no effect on lesion size or composition. Taken together, these results show that IL-1ß has multiple beneficial effects in late-stage murine atherosclerosis, including promotion of outward remodeling and formation and maintenance of an SMC- and collagen-rich fibrous cap.

Defining the functional network of epigenetic regulators in Arabidopsis thaliana.

Development of ChIP-chip and ChIP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting (CC) Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator's expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.