Mark R. Heckle

The Transcription Factor CREB Enhances Interleukin-17A Production and Inflammation in a Mouse Model of Atherosclerosis

A lipoxygenase exacerbates atherosclerosis in mice by stimulating macrophages to produce a proinflammatory cytokine. Lipoxygenase, CREB, and Atherosclerosis The chronic inflammatory disease atherosclerosis is characterized by thickening of the arterial wall through the accumulation of lipid-laden foam cells derived from macrophages and smooth muscle cells. It is thought that lipoxygenases (LOs), which metabolize polyunsaturated fatty acids, play key roles in the pathogenesis of atherosclerosis by oxidizing low-density lipoprotein (LDL). Kotla et al. found that the major 12/15-LO product in mice, 15(S)-HETE, stimulated the production of reactive oxygen species in monocytes and macrophages, which culminated in production of the proinflammatory cytokine interleukin-17A (IL-17A) in a manner dependent on the transcription factor CREB. Loss of the gene encoding 12/15-LO in a mouse model of atherosclerosis resulted in decreased accumulation of macrophages at atherosclerotic lesions, decreased fat deposits, and reduced abundance of IL-17A. Together, these data suggest that 12/15-LO exacerbates atherosclerosis in vivo by stimulating the CREB-dependent production of IL-17A and enhancing inflammation. The enzyme 15-lipoxygenase (15-LO) plays a role in atherogenesis (also known as atherosclerosis), but the underlying mechanisms are unclear. We found that 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO–dependent metabolite of arachidonic acid, stimulated the production of reactive oxygen species (ROS) by monocytes through the xanthine oxidase–mediated activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. ROS production led to the Syk-, Pyk2-, and mitogen-activated protein kinase (MAPK)–dependent production of the proinflammatory cytokine interleukin-17A (IL-17A) in a manner that required the transcription factor CREB (cyclic adenosine monophosphate response element–binding protein). In addition, this pathway was required for the 15(S)-HETE–dependent migration and adhesion of monocytes to endothelial cells. Consistent with these observations, we found that peritoneal macrophages from apolipoprotein E–deficient (ApoE−/−) mice fed a high-fat diet (a mouse model of atherosclerosis) exhibited increased xanthine oxidase and NADPH oxidase activities; ROS production; phosphorylation of Syk, Pyk2, MAPK, and CREB; and IL-17A production compared to those from similarly fed ApoE−/−:12/15-LO−/− mice. These events correlated with increased lipid deposits and numbers of monocytes and macrophages in the aortic arches of ApoE−/− mice, which resulted in atherosclerotic plaque formation. Together, these observations suggest that 15(S)-HETE exacerbates atherogenesis by enhancing CREB-dependent IL-17A production and inflammation.

Protein Kinase N1 Is a Novel Substrate of NFATc1-mediated Cyclin D1-CDK6 Activity and Modulates Vascular Smooth Muscle Cell Division and Migration Leading to Inward Blood Vessel Wall Remodeling

Background: The purpose of this study was to test the role of PKN1 in vascular wall remodeling. Results: PKN1 mediates MCP-1-induced HASMC migration/proliferation and balloon injury-induced neointima formation. Conclusion: PKN1 plays a role in vascular wall remodeling following balloon injury. Significance: PKN1 could be a promising target for the next generation of drugs for vascular diseases such as restenosis. Toward understanding the mechanisms of vascular wall remodeling, here we have studied the role of NFATc1 in MCP-1-induced human aortic smooth muscle cell (HASMC) growth and migration and injury-induced rat aortic wall remodeling. We have identified PKN1 as a novel downstream target of NFATc1-cyclin D1/CDK6 activity in mediating vascular wall remodeling following injury. MCP-1, a potent chemoattractant protein, besides enhancing HASMC motility, also induced its growth, and these effects require NFATc1-dependent cyclin D1 expression and CDK4/6 activity. In addition, MCP-1 induced PKN1 activation in a sustained and NFATc1-cyclin D1/CDK6-dependent manner. Furthermore, PKN1 activation is required for MCP-1-induced HASMC growth and migration. Balloon injury induced PKN1 activation in NFAT-dependent manner and pharmacological or dominant negative mutant-mediated blockade of PKN1 function or siRNA-mediated down-regulation of its levels substantially suppressed balloon injury-induced smooth muscle cell migration and proliferation resulting in reduced neointima formation. These novel findings suggest that PKN1 plays a critical role in vascular wall remodeling, and therefore, it could be a promising new target for the next generation of drugs for vascular diseases, particularly restenosis following angioplasty, stent implantation, or vein grafting.

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

Background: The major goal of this study is to test the hypothesis that thrombin plays a role in inflammation. Results: Thrombin stimulates monocyte F-actin cytoskeletal remodeling and migration by PAR1, Gα12, Pyk2, Gab1, Rac1, and RhoA-dependent Pak2 activation. Conclusion: Pak2 mediates thrombin-PAR1-induced monocyte/macrophage migration. Significance: PAR1 could be a potential target for the development of anti-inflammatory drugs. To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα12 negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation.

Novel Role of Proline-Rich Nonreceptor Tyrosine Kinase 2 in Vascular Wall Remodeling After Balloon Injury

Objective—To investigate the role of Pyk2, a proline-rich nonreceptor tyrosine kinase, in G protein−coupled receptor agonist, thrombin-induced human aortic smooth muscle cell growth and migration, and injury-induced vascular wall remodeling. Methods and Results—Thrombin, a G protein–coupled receptor agonist, activated Pyk2 in a time-dependent manner and inhibition of its stimulation attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. Thrombin also activated Grb2-associated binder protein 1, p115 Rho guanine nucleotide exchange factor, Rac1, RhoA, and p21-activated kinase 1 (Pak1) and interference with stimulation of these molecules attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. In addition, adenovirus-mediated expression of dominant negative Pyk2 inhibited thrombin-induced Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA and Pak1 stimulation. Balloon injury also caused activation of Pyk2, Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA, and Pak1 in the carotid artery of rat, and these responses were sensitive to inhibition by the dominant negative Pyk2. Furthermore, inhibition of Pyk2 activation resulted in reduced recruitment of smooth muscle cells onto the luminal surface and their proliferation in the intimal region leading to suppression of neointima formation. Conclusion—Together, these results demonstrate for the first time that Pyk2 plays a crucial role in G protein–coupled receptor agonist thrombin-induced human aortic smooth muscle cell growth and migration, as well as balloon injury–induced neointima formation.

Myofibroblast secretome and its auto-/paracrine signaling.

ABSTRACT Myofibroblasts (myoFb) are phenotypically transformed, contractile fibroblast-like cells expressing α-smooth muscle actin microfilaments. They are integral to collagen fibrillogenesis with scar tissue formation at sites of repair irrespective of the etiologic origins of injury or tissue involved. MyoFb can persist long after healing is complete, where their ongoing turnover of collagen accounts for a progressive structural remodeling of an organ (a.k.a. fibrosis, sclerosis or cirrhosis). Such persistent metabolic activity is derived from a secretome consisting of requisite components in the de novo generation of angiotensin (Ang) II. Autocrine and paracrine signaling induced by tissue AngII is expressed via AT1 receptor ligand binding to respectively promote: i) regulation of myoFb collagen synthesis via the fibrogenic cytokine TGF-β1-Smad pathway; and ii) dedifferentiation and protein degradation of atrophic myocytes immobilized and ensnared by fibrillar collagen at sites of scarring. Several cardioprotective strategies in the prevention of fibrosis and involving myofibroblasts are considered. They include: inducing myoFb apoptosis through inactivation of antiapoptotic proteins; AT1 receptor antagonist to interfere with auto-/paracrine myoFb signaling or to induce counterregulatory expression of ACE2; and attacking the AngII-AT1R-TGF-β1-Smad pathway by antibody or the use of triplex-forming oligonucleotides.

Racial Difference in Symptom Onset to Door Time in ST Elevation Myocardial Infarction

Background There are poorer outcomes following ST elevation myocardial infarction in blacks compared to white patients despite comparable door‐to‐reperfusion time. We hypothesized that delays to hospital presentation may be contributory. Methods and Results We conducted a retrospective analysis of the 1144 patients admitted for STEMI in our institution from 2008 to 2013. The door‐to‐balloon time (D2BT) and symptom‐onset‐to‐door time (SODT) were compared by race. Bivariate analysis was done comparing the median D2BT and SODT. Stratified analyses were done to evaluate the effect of race on D2BT and SODT, accounting for insurance status, age, sex and comorbidities. The mean age was 59±13 years; 56% of this population was black and 41% was white. Males accounted for 66% of this population. The median D2BT was 60 minutes (interquartile range [IQR] 42–82), and median SODT was 120 minutes (IQR 60–720). There was no significant difference in D2BT by race (P=0.86). Black patients presented to the emergency room (ER) later than whites (SODT=180 [IQR 60–1400] vs 120 [IQR 60–560] minutes, P<0.01) and were more likely to be uninsured (P<0.01). After controlling for comorbidities, insurance, and socioeconomic status, blacks were 60% more likely to present late after a STEMI (OR 1.6, P<0.01). A subset analysis excluding transferred patients showed similar results. Conclusions Black patients present later to the ER after STEMI with no difference in D2BT compared to whites. This difference in time to presentation may be one of the factors accounting for poor outcomes in this population.

Atrophied cardiomyocytes and their potential for rescue and recovery of ventricular function

Cardiomyocytes must be responsive to demands placed on the heart’s contractile work as a muscular pump. In turn, myocyte size is largely dependent on the workload they perform. Both hypertrophied and atrophic myocytes are found in the normal and diseased ventricle. Individual myocytes become atrophic when encumbered by fibrillar collagen, such as occurs at sites of fibrosis. The mechanisms include: (a) being immobilized and subject to disuse with ensuing protein degradation mediated by redox-sensitive, proteolytic ligases of the ubiquitin–proteasome system and (b) dedifferentiated re-expressing fetal genes induced by low intracellular triiodothyronine (T3) via thyroid hormone receptor β1. This myocyte-selective, low T3 state is a consequence of heterocellular signaling emanating from juxtaposed scar tissue myofibroblasts and their secretome with its de novo generation of angiotensin II. In a paracrine manner, angiotensin II promotes myocyte Ca2+ entry and subsequent Ca2+ overload with ensuing oxidative stress that overwhelms antioxidant defenses to activate deiodinase-3 and its enzymatic degradation of T3. In the failing heart, atrophic myocytes represent an endogenous population of viable myocytes which could be rescued to augment contractile mass, reduce systolic wall stress (afterload) and recover ventricular function. Experimental studies have shown the potential for the rescue and recovery of atrophic myocytes in rebuilding the myocardium—a method complementary to today’s quest in regenerating myocardium using progenitor cells.

Cannabinoids and Symptomatic Bradycardia

ABSTRACT Cannabinoids, the bioactive components of marijuana, have adverse cardiovascular consequences, including symptomatic sinus bradycardia, sinus arrest and ventricular asystole. Physicians should be aware of these deleterious consequences which can appear in otherwise healthy persons who are chronic marijuana users.

Multifactorial Brugada Phenocopy—Reply

Multifactorial Brugada Phenocopy To the Editor We read with interest the article by Heckle et al1 recently published in the Challenges in Clinical Electrocardiography section of JAMA Internal Medicine. The authors addressed the case of a woman in her 50s who presented with altered mentalstatusandrespiratorydistressrequiringintubationinthefield. Anelectrocardiogram(ECG)wasrecordedonpresentation,which was interpreted by the authors as a normal sinus rhythm with a right bundle branch pattern and loss of P-wave amplitude in the precordial leads. It was pointed out that the most profound ECG abnormality was the greater than 5-mm coved-type ST-segment elevation in leads V1 to V2 with the presence of Q waves. After decreasing the potassium level, the ECG no longer presented segment elevation in leads V1 to V2. The authors attributed the ST-segment changes to hyperkalemia in the setting of rhabdomyolysis, acute renal failure, acidosis, and angiotensin-converting enzyme inhibitor use. In our opinion, the ECG showed in leads V1 to V2 a typical Brugada type-1 pattern with 2 mm or more coved ST-segment elevation followed by a negative T-wave. Considering this, it would have been desirable to perform, once hyperkalemia resolved, a provocative challenge with a sodium channel blocker. If negative, the diagnosis to consider is Brugada phenocopy.2 Brugada phenocopies are clinical entities with identical electrocardiographic patterns as true congenital Brugada syndrome, but caused by various clinical circumstances. There are several factors that we would like to discuss and that could have played an important role in the development of this pattern. Hyperkalemia, among others, has been identified as a metabolic condition that can reproduce the BS pattern by decreasing the resting membrane potential, which determines an inactivation of the sodium channels. This produces an imbalance between the entrance of sodium to the cell and the exit of potassium with predominance of the latter which is more pronounced in the right ventricle and more active in the epicardial cells than in the endocardium and in the M cells.3 Another factor that was not taken into account by the authors was the intake of amitriptyline. This tricyclic antidepressant can reproduce the Brugada ECG pattern both in clinical practice and in experimental models.4 On the other hand, it is well known that cocaine blocks the sodium channels and therefore could have some role in unmasking true Brugada syndrome.5 We invite the authors to send their c ase to our international registry, which can be found at http://www .brugadaphenocopy.com.

Accurate Prediction of False ST-Segment Elevation Myocardial Infarction: Ready for Prime Time?

The incidence of inappropriate cardiac catheterization lab activation for treatment of a false ST-segment elevation myocardial infarction (STEMI) has been reported to be 2.6%-36%. Excessive inappropriate catheterization lab activation may be associated with risks to patients, provider fatigue and improper resource usage. HYPOTHESIS To derive and validate a prediction score to more accurately classify patients with STEMI. METHODS AND RESULTS We conducted a retrospective cohort analysis of 1144 consecutive patients initially diagnosed with STEMI between September 2008 and January 2013. The incidence of catheterization laboratory activation for false STEMI was 21.4%. Multiple logistic regression identified 8 factors as important for prediction of false STEMI. Using a prediction rule derived from these factors, the area under the curve for differentiating false from true STEMI patients was 0.80 (95% CI: 0.75-0.84). Using objective standards, criteria were defined that had 95% specificity for detecting patients with an incorrect diagnosis of STEMI. IN CONCLUSION A prediction rule has been derived and validated in a large, racially diverse group to identify false STEMI patients with an incorrect classification rate of 5%, which is an improvement over current clinical practice. Prediction rules may be particularly useful in patients with atypical presentations in which emergent catheterization cannot be achieved rapidly or carries significant patient risk.