Scott J. Cameron

Antibody to human leukocyte antigen triggers endothelial exocytosis.

Although antibodies to HLA play a role in the pathogenesis of diseases processes such as rejection of transplanted organs, the precise mechanisms by which antibodies cause tissue injury are not completely understood. We hypothesized that antibodies to host tissues cause inflammation in part by activating endothelial exocytosis of granules that contain prothrombotic mediators such as von Willebrand Factor (VWF) and proinflammatory mediators such as P-selectin. To test this hypothesis, we treated human endothelial cells with murine monoclonal antibody W6/32 to HLA class I and then measured exocytosis by the release of VWF and the externalization of P-selectin. Antibody to HLA activates endothelial exocytosis in a dose-dependent manner over time. The biologically active complement split product, C5a, adds a slight but significant increase to antibody induction of exocytosis. Antibody to HLA alone or with C5a did not damage the cells. Cross-linking of HLA appears to play a role in the ability of antibody to activate exocytosis, because the W6/32 monovalent Fab fragment did not activate VWF release, but the bivalent F(ab′)2 was effective in triggering exocytosis. To explore the in vivo effects of antibody upon graft injury, we infused W6/32 F(ab′)2 antibody to human HLA into severe combined immunodeficient/beige mice that had been transplanted with human skin grafts. Antibody to HLA activated exocytosis and inflammation in human skin grafts. Our data show that antibody to host antigens can activate human endothelial cell exocytosis and leukocyte trafficking. By triggering vascular inflammation, antibody activation of exocytosis may play a role in transplant rejection.

Regulation of Epidermal Growth Factor-induced Connexin 43 Gap Junction Communication by Big Mitogen-activated Protein Kinase 1/ERK5 but Not ERK1/2 Kinase Activation

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5α, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.

HMG-CoA Reductase Inhibitors Inhibit Endothelial Exocytosis and Decrease Myocardial Infarct Size

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors protect the vasculature from inflammation and atherosclerosis by cholesterol dependent and cholesterol independent mechanisms. We hypothesized that HMG-CoA reductase inhibitors decrease exocytosis of Weibel-Palade bodies, endothelial cell granules whose contents promote thrombosis and vascular inflammation. We pretreated human aortic endothelial cells with simvastatin for 24 hours, then stimulated the cells with thrombin, and measured the amount of vWF released into the media. We then measured the effect of simvastatin on myocardial infarction in mice. Simvastatin decreased thrombin-stimulated Weibel-Palade body exocytosis by 89%. Simvastatin inhibited exocytosis in part by increasing synthesis of nitric oxide (NO), which S-nitrosylated N-ethylmaleimide sensitive factor (NSF), a critical regulator of exocytosis. Simvastatin treatment attenuated myocardial infarct size by 58% in wild-type but not eNOS knockout mice. Furthermore, simvastatin decreased endothelial exocytosis and neutrophil infiltration into ischemic-reperfused myocardium, which was mediated in part by P-selectin contained in Weibel-Palade bodies. However, simvastatin did not affect exocytosis and inflammation in myocardial infarcts of eNOS knockout mice. Inhibition of endothelial exocytosis is a novel mechanism by which HMG-CoA reductase inhibitors may reduce vascular inflammation, inhibit thrombosis, and protect the ischemic myocardium. These findings may explain part of the pleiotropic effects of statin therapy for patients with cardiovascular disease.

Aldosterone activates endothelial exocytosis.

Although elevated levels of aldosterone are associated with vascular inflammation, the proinflammatory pathways of aldosterone are not completely defined. We now show that aldosterone triggers endothelial cell exocytosis, the first step in leukocyte trafficking. Exogenous aldosterone stimulates endothelial exocytosis of Weibel-Palade bodies, externalizing P-selectin and releasing von Willebrand factor. Spironolactone, a nonselective mineralocorticoid receptor (MR) blocker, antagonizes aldosterone-induced endothelial exocytosis. Knockdown of the MR also decreases exocytosis, suggesting that the MR mediates exocytosis. Aldosterone triggers exocytosis within minutes, and this effect is not inhibited by actinomycin D, suggesting a nongenomic effect of aldosterone. Aldosterone treatment of endothelial cells increases leukocyte adherence to endothelial cells in culture. Taken together, our data suggest that aldosterone activates vascular inflammation in part through nongenomic, MR-mediated pathways. Aldosterone antagonism may decrease vascular inflammation and cardiac fibrosis in part by blocking endothelial exocytosis.

Activation of big MAP kinase 1 (BMK1/ERK5) inhibits cardiac injury after myocardial ischemia and reperfusion.

Big MAP kinase 1 (BMK1/ERK5) plays a critical role in pre‐natal development of the cardiovascular system and post‐natal eccentric hypertrophy of the heart. Of the two isoforms upstream of MAPK‐kinase 5 (MEK5) known to exist, only the longer MEK5α isoform potently activates BMK1. We generated cardiac‐specific constitutively active form of the MEK5α (CA‐MEK5α transgenic (Tg) mice), and observed a 3 to 4‐fold increase in endogenous BMK1 activation and hyperphosphorylation of connexin 43 in the ventricles of the Tg compared to wild‐type mice. The CA‐MEK5α‐Tg‐mice demonstrated a profoundly accelerated recovery of left ventricular developed pressure after ischemia/reperfusion. We propose a novel role for BMK1 in protecting the heart from ischemia/reperfusion‐induced cardiac injury.

Essential role for verotoxin in sustained stress-activated protein kinase and nuclear factor kappa b signaling, stimulated by escherichia coli o157:H7 in vero cells

ABSTRACT The effects of Escherichia coli O157:H7 (strains E30480 and PM601) and the associated verotoxins (VTs), VT1 and VT2, on stress-activated protein kinase and nuclear factor kappa B (NF-κB) signaling were investigated with Vero cells, which are extremely sensitive to the cytotoxic effects of E. coli O157:H7 in vitro. Cell-free supernatants prepared from E30480 and PM601 cultures and purified VT1 and VT2 stimulated a strong and prolonged (>4-h) activation of both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. However, JNK activity stimulated in response to E30480 supernatants was substantially reduced following pretreatment with anti-VT1 and anti-VT2 antibodies, while a VT1 and VT2 gene knockout mutant of PM601 was unable to stimulate JNK activity. E30480 supernatants also caused a sustained activation of NF-κB DNA binding, degradation of inhibitory kappa B alpha (IκBα), and an increase in inhibitory kappa B kinase α activity, although PM601 supernatants and VT1 and VT2 had no effect. However, preincubation with VTs prolonged the transient activation of NF-κB and IκBα degradation stimulated by either tumor necrosis factor alpha or interleukin 1β, while preincubation with anti-VT antibodies prevented the prolonged loss of IκBα and partially reduced DNA binding in response to E30480 supernatants. These results strongly suggest that in Vero cells, VT plays an essential role in sustained JNK and NF-κB signaling in response to E. coli O157:H7 and that this action may underpin their cell-selective cytotoxic effects. These studies also suggest that another component released by strain E30480 contributes to the early activation of JNK and NF-κB.

Pre-Analytic Considerations for the Proper Assessment of Hormones of the Hypothalamic-Pituitary Axis in Epidemiological Research

There is growing epidemiological interest in hormones as predictors of chronic diseases. The correct handling and analysis of hormones can be cumbersome, and great care must be taken in these processes in order to gain the most information possible. Given differences in sampling, processing, and stability of the various hormonal assays, we sought to provide a comprehensive review to aid future epidemiological research. We have coupled a thorough literature search with our own analytical experience to outline common laboratory problems one must consider in analyzing the hormones of the hypothalamic-pituitary axis. In addition, we describe the benefits and limitations of using alternative media – including urine, saliva, and blood spots on filter paper – to measure endocrine hormones in epidemiological studies.

High-density lipoprotein metabolism and endothelial function.

Purpose of reviewHigh-density lipoprotein (HDL) protects against atherosclerosis, transporting cholesterol from peripheral cells to the liver, where it is excreted into the bile. However, HDL also has prominent vascular protective effects. Recent findingsRecent studies have uncovered mechanisms through which HDL decreases vascular inflammation, boosts nitric oxide production, and inhibits thrombosis. The discovery that dysfunctional HDL can also have proinflammatory effects has uncovered a new aspect of HDL biology. SummaryLow-density lipoprotein is the primary target for drug therapy of dyslipidemias. Drugs that increase HDL also affect additional metabolic pathways. Development of selective drugs targeting key aspects of HDL metabolism may enable us to alter the composition of HDL and inhibit atherogenesis.

Pseudohyponatremia in a patient with HIV and hepatitis C coinfection.

Pseudohyponatremia refers to low serum sodium in the presence of normal plasma tonicity. Whereas pseudohyponatremia secondary to hyperlipidemia is a commonly recognized occurrence, falsely low sodium levels secondary to elevated protein are less frequently observed. We present in this paper the case of a man coinfected with HIV and hepatitis C who had pseudohyponatremia from hypergammaglobulinemia. As hypergammaglobulinemia is a frequent occurrence in both HIV and HCV, we suggest that pseudohyponatremia is an important and likely underdiagnosed phenomenon in this patient population. Clinicians need to be aware of the electrolyte exclusion effect and become familiar with the techniques used by their local laboratory in the measurement of serum electrolytes. Pseudohyponatremia should also be included in the differential diagnosis of an elevated osmolal gap, as the falsely lowered sodium level will lead to a falsely low calculated serum osmolality.

Platelet Extracellular Regulated Protein Kinase 5 Is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion.

Background— Platelets have a pathophysiologic role in the ischemic microvascular environment of acute coronary syndromes. In comparison with platelet activation in normal healthy conditions, less attention is given to mechanisms of platelet activation in diseased states. Platelet function and mechanisms of activation in ischemic and reactive oxygen species–rich environments may not be the same as in normal healthy conditions. Extracellular regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase family member activated in hypoxic, reactive oxygen species–rich environments and in response to receptor-signaling mechanisms. Prior studies suggest a protective effect of ERK5 in endothelial and myocardial cells after ischemia. We present evidence that platelets express ERK5 and that platelet ERK5 has an adverse effect on platelet activation via selective receptor-dependent and receptor-independent reactive oxygen species–mediated mechanisms in ischemic myocardium. Methods and Results— Using isolated human platelets and a mouse model of myocardial infarction (MI), we found that platelet ERK5 is activated post-MI and that platelet-specific ERK5–/– mice have less platelet activation, reduced MI size, and improved post-MI heart function. Furthermore, the expression of downstream ERK5-regulated proteins is reduced in ERK5–/– platelets post-MI. Conclusions— ERK5 functions as a platelet activator in ischemic conditions, and platelet ERK5 maintains the expression of some platelet proteins after MI, leading to infarct expansion. This demonstrates that platelet function in normal healthy conditions is different from platelet function in chronic ischemic and inflammatory conditions. Platelet ERK5 may be a target for acute therapeutic intervention in the thrombotic and inflammatory post-MI environment.