Timothy J. Stalker

Inhibition of Rho-Kinase Leads to Rapid Activation of Phosphatidylinositol 3-Kinase/Protein Kinase Akt and Cardiovascular Protection

Objective—Rho-Kinase activity is increased in cardiovascular diseases and in patients with cardiovascular risk factors. However, it is not known whether inhibition of Rho-kinase could lead to cardiovascular protection and, if so, by what mechanism. Methods and Results—In human endothelial cells, the Rho-kinase inhibitor, hydroxyfasudil (HF) (1 to 100 &mgr;mol/L), increased Akt serine-473 phosphorylation within 15 minutes, leading to a 2.2-fold and 4.0-fold increase in Akt kinase activity and nitric oxide (NO) release, respectively. Activation of Akt and eNOS by HF was completely blocked by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002 (10 &mgr;mol/L). To determine the physiological relevance of this pathway, we used 2 models of ischemia-reperfusion (I/R) injury. Acute administration of fasudil (10 mg/kg, intraperitoneal, 1 hour before ischemia) decreased leukocyte recruitment and adhesion to the mesenteric endothelium after I/R injury in wild-type but not eNOS−/− mice. Similarly, treatment with fasudil decreased myocardial infarct size by 38% in rats subjected to transient coronary artery occlusion. Cotreatment with 2 PI3-kinase inhibitors, wortmannin and LY294002, or the eNOS inhibitor, l-NAME, blocked the cardiovascular protective effects of fasudil. Conclusions—Inhibition of Rho-kinase leads to the activation of the PI3-kinase/Akt/eNOS pathway and cardiovascular protection. These findings suggest that Rho-kinase may play an important role in mediating the inflammatory response to I/R injury.

A new HMG-CoA reductase inhibitor, rosuvastatin, exerts anti-inflammatory effects on the microvascular endothelium: the role of mevalonic acid.

Recent studies have reported that hydroxymethylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors have vasculoprotective effects independent of their lipid‐lowering properties, including anti‐inflammatory actions. We used intravital microscopy of the rat mesenteric microvasculature to examine the effects of rosuvastatin, a new HMG‐CoA reductase inhibitor, on leukocyte‐endothelium interactions induced by thrombin. Intraperitoneal administration of 0.5 and 1.25 mg kg−1 rosuvastatin 18 h prior to the study, significantly and dose‐dependently attenuated leukocyte rolling, adherence, and transmigration in the rat mesenteric microvasculature superfused with 0.5 u ml‐1 thrombin. This protective effect of rosuvastatin was reversed by intraperitoneal injection of 25 mg kg−1 mevalonic acid 18 h before the study. Immunohistochemical detection of the endothelial cell adhesion molecule P‐selectin showed a 70% decrease in endothelial cell surface expression of P‐selectin in thrombin‐stimulated rats given 1.25 mg kg−1 rosuvastatin. In addition, rosuvastatin enhanced release of nitric oxide (NO) from the vascular endothelium as measured directly in rat aortic segments. Moreover, rosuvastatin failed to attenuate leukocyte‐endothelium interactions in peri‐intestinal venules of eNOS−/− mice. These data indicate that rosuvastatin exerts important anti‐inflammatory effects via inhibition of endothelial cell adhesion molecule expression, and that this protective action of rosuvastatin requires release of nitric oxide by the vascular endothelium. These data also demonstrate that the mechanism of the non‐lipid lowering actions of HMG‐CoA reductase inhibitors in vivo may be due to reduced formation or availability of mevalonic acid within endothelial cells.

Regulated surface expression and shedding support a dual role for semaphorin 4D in platelet responses to vascular injury.

Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, CD72 and plexin-B1. Soluble sema4D has been shown to evoke angiogenic responses from endothelial cells and impair monocyte migration, but the origin of soluble sema4D, particularly at sites of vascular injury, has been unclear. Here we show that platelets express sema4D and both of its receptors and provide evidence that these molecules promote thrombus formation. We also show that the surface expression of sema4D and CD72 increases during platelet activation, followed by the gradual shedding of the sema4D extracellular domain. Shedding is blocked by metalloprotease inhibitors and abolished in mouse platelets that lack the metalloprotease ADAM17 (TACE). Mice that lack sema4D exhibit delayed arterial occlusion after vascular injury in vivo, and their platelets show impaired collagen responses in vitro. In resting platelets, as in B lymphocytes, CD72 is associated with the protein tyrosine phosphatase SHP-1. Platelet activation causes dissociation of the complex, as does the addition of soluble sema4D. These findings suggest a dual role for sema4D in vascular responses to injury. As thrombus formation begins, platelet-associated sema4D can bind to its receptors on nearby platelets, promoting thrombus formation. As thrombus formation continues, sema4D is shed from the platelet surface and becomes available to interact with receptors on endothelial cells and monocytes, as well as continuing to interact with platelets.

Minding the gaps to promote thrombus growth and stability

Efforts to understand the role of platelets in hemostasis and thrombosis have largely focused on the earliest events of platelet activation, those that lead to aggregation. Although much remains to be learned about those early events, this Review examines a later series of events: the interactions between platelets that can only occur once aggregation has begun, bringing platelets into close contact with each other, creating a protected environment in the gaps between aggregated platelets, and fostering the continued growth and stability of the hemostatic plug.

A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbβ3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.

A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets.

Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth.

Myocardial Ischemia/Reperfusion Injury in NADPH Oxidase–Deficient Mice

Previous studies have suggested that oxygen-derived free radicals are involved in the pathophysiology of myocardial ischemia/reperfusion (MI/R) injury. Specifically, neutrophils have been shown to mediate postischemic ventricular arrhythmias and myocardial necrosis. We hypothesized that MI/R injury would be reduced in the absence (-/-) of NADPH oxidase. Heterozygous control mice (n=23) and NADPH oxidase(-/-) mice (n=24) were subjected to 30 minutes of coronary artery occlusion and 24 hours of reperfusion. Myocardial area at risk per left ventricle was similar in heterozygous control hearts (55+/-3%) and NADPH oxidase(-/-) hearts (61+/-4%). Contrary to our hypothesis, the size of infarct area at risk was similar in the heterozygous control mice (42+/-4%) and NADPH oxidase(-/-) mice (34+/-5%) (P=not significant). In addition, echocardiographic examination of both groups revealed that left ventricle fractional shortening was similar in NADPH oxidase(-/-) mice (n=8; 27+/-2.5%) and heterozygous control mice (n=10; 23.3+/-3. 3%) after MI/R. Superoxide production, as detected by cytochrome c reduction, was significantly impaired (P<0.01) in NADPH oxidase(-/-) mice (n=6) compared with heterozygous mice (n=7) (0.04+/-0.03 versus 2.2+/-0.08 nmol O(2).min(-1).10(6) cells(-1)). Intravital microscopy of the inflamed mesenteric microcirculation demonstrated that leukocyte rolling and adhesion were unaffected by the absence of NADPH oxidase. Oyster glycogen-stimulated neutrophil transmigration into the peritoneum was also similar in both the heterozygous control mice and NADPH oxidase(-/-) mice (P:=not significant). These findings suggest that NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after MI/R.

A novel role for calpains in the endothelial dysfunction of hyperglycemia

Recent studies have reported that the activity of the calcium‐dependent protease calpain is increased in acute inflammatory processes of the cardiovascular system. Because diabetes is associated with vascular inflammation, we hypothesized that increased calpain activity in response to hyperglycemia may play a role in diabetic cardiovascular disease. The effects of calpain inhibition on leukocyte‐endothelium interactions induced by hyperglycemia were examined by intravital microscopy. Intraperitoneal administration of the selective calpain inhibitor benzyloxycarbonyl‐leucyl‐leucinal (5 µmol/L) prevented the up‐regulation of leukocyte‐endothelium interactions in response to 25 mmol/L d‐glucose via a nitric oxide‐dependent mechanism. Furthermore, treatment of rats with d‐glucose significantly decreased basal endothelial NO release in mesenteric post‐capillary venules, a phenomenon prevented by inhibition of calpain activity. Immunoprecipitation studies revealed that glucose induces loss of NO via a calpain‐dependent decrease in the association of hsp90 with endothelial nitric oxide synthase. In addition, inhibition of calpain activity decreased endothelial cell surface expression of the pro‐inflammatory adhesion molecules ICAM‐1 and VCAM‐1 during hyperglycemia. These data demonstrate that calpains contribute to important inflammatory events during hyperglycemia and that pharmacological inhibition of calpain activity attenuates leukocyte‐endothelium interactions and preserves eNOS function.

Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse

Summary.  Background:  Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics.

A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment

Hemostatic thrombi formed after a penetrating injury have a heterogeneous architecture in which a core of highly activated, densely packed platelets is covered by a shell of less-activated, loosely packed platelets. In the first manuscript in this series, we show that regional differences in intrathrombus protein transport rates emerge early in the hemostatic response and are preserved as the thrombus develops. Here, we use a theoretical approach to investigate this process and its impact on agonist distribution. The results suggest that hindered diffusion, rather than convection, is the dominant mechanism responsible for molecular movement within the thrombus. The analysis also suggests that the thrombus core, as compared with the shell, provides an environment for retaining soluble agonists such as thrombin, affecting the extent of platelet activation by establishing agonist-specific concentration gradients radiating from the site of injury. This analysis accounts for the observed weaker activation and relative instability of platelets in the shell and predicts that a failure to form a tightly packed thrombus core will limit thrombin accumulation, a prediction tested by analysis of data from mice with a defect in clot retraction.