- Email: [email protected]
Vascular device interaction with the endothelium
Abstracts / Biomedicine & Pharmacotherapy 62 (2008) 503e512
504
shear stress, whereas susceptible regions, e.g. arches and branches, are exposed to low / oscillatory shear. Shear stress regulates vascular inflammation and atherosclerosis by altering the physiology of endothelial cells (EC), which detect shear stress via a tri-molecular complex expressed at their surface. High shear stress suppresses EC activation by inhibiting pro-inflammatory NF-kB and MAP kinase e AP-1 signaling via mechanisms that involve the activation of KLF2 and Nrf2 transcription factors. In contrast, low and oscillatory shear enhances endothelial activation via activation of MAP kinase e AP-1 and NF-kB pathways. These mechanisms are likely to contribute to the focal distribution of atherosclerotic lesions and could potentially be exploited to design novel therapeutic strategies to protect susceptible regions of the arterial tree from inflammation and atherosclerosis. E-mail address: [email protected]
3 Leukocyte-Endothelial Interactions with Respect to the Adhesion Cascade Dr Victoria Ridger Cardiovascular Research Unit, School of Medicine and Biomedical Sciences,University of Sheffield, UK Leukocyte recruitment to the site of injury and inflammation occurs via a sequence highly regulated events, collectively known as the adhesion cascade. They are characterised as capture, rolling, slow rolling, crawling, adhesion and extravasation. At each step, leukocytes interact with the endothelium through adhesion molecules; the selectins and their ligands regulate the first contact events and rolling whereas the integrins are involved in the later stages. Not only do these molecules control the physical interactions between leukocytes and endothelial cells, but they also are involved in signalling and preparing the leukocyte for the subsequent steps in the cascade. The onset of inflammation increases the expression and alters the avidity of these adhesion molecules on both the leukocytes and the endothelium, increasing the adhesive environment. Marginated leukocytes make initial contact with endothelial cells through interaction of L- (on leukocyte) or P- (on endothelial cells) selectins with their respective ligands. Rolling then occurs through a series of bond formations at the leading edge and breakages at the trailing edge of the leukocyte. The next stage of the cascade is slow rolling, mediated by E-selectin, observed after longer periods of endothelial stimulation. Slow rolling is thought to play a role in prolonging the exposure of rolling leukocytes to chemokines. Rolling also triggers cell signalling events thought to prepare the cell for the next step of ‘‘firm’’ adhesion. Chemokines are involved in the transition of rolling into firm adhesion and in the shedding of L-selectin from the surface of leukocytes. Firm adhesion is known to be highly dependent on b2 integrins and their ligands, members of the immunoglobulin superfamily. Once firmly adherent, leukocytes crawl towards endothelial-endothelial cell junctions via these same interactions. Transendothelial migration is
generally thought to occur in an intercellular manner and is mediated by integrin PECAM-1, ICAM-2, JAM-A and CD99. However this latter stage is not as well characterised. The understanding of the adhesion cascade and the multiple steps involved in leukocyte recruitment is essential for the development of effective, novel therapies for inflammatory diseases. E-mail address: [email protected]
4 Vascular device interaction with the endothelium Mark Atherton 1, Ashraf W Khir 1, Marco Cavazzuti 2, Giovanni Barozzi 2, & Michael Collins 1 1
School of Engineering and Design, Brunel University, West London, UK 2 Dipartminto di Ingegneria Meccanica e Civile, Universita` di Modena e Reggio Emilia, Italy Cerebral stents and Intra Aortic Balloon Pumps (IABP) are examples of mechanical devices that are inserted into arteries to restore flows to clinically healthy states. The stent and the IABP ‘correct’ the arterial flow by static dilation and by cyclical occlusion respectively. As this presentation shows, these functions are effectively modelled by current engineering practice. As interventions however, by their very nature they involve physical contact between a non-biological structure and the sensitive endothelial surface. The possible damage to the endothelium is not currently well addressed and we also consider this issue. Cerebral stents generally have two primary clinical objectives: to mechanically dilate a stenosed artery and to have minimal detrimental impact upon local blood flow characteristics. These objectives are well served at the arterial scale as these devices are evidently effective in opening up diseased arteries and restoring vital flows. However, at the near-wall micro-scale the picture is less satisfactory, as thin stent wires apply stresses to the endothelium and glycocalyx and the local flow is disturbed rather than being ideally streamlined. This causes further interaction with this endothelium topography. Wall Shear Stress (WSS) is the measure commonly used to indicate the interaction between fluid and wall but it is a broad brush approach that loses fidelity close to the wall. We will present simulation results of blood flow through a stented cerebral saccular aneurysm under these limitations of WSS. The Intra Aortic Balloon Pump (IABP) is a widely used temporary cardiac assist device. The balloon is usually inserted from the iliac artery, advanced in the aorta until it reaches the desired position; with its base just above the renal bifurcation and the tip approximately 10cm away from the aortic valve. The balloon is inflated and deflated every(1:1), every other- (1:2) or every second (1:3) cardiac cycle. Balloon inflation, which takes place during early diastole, causes an increase in the pressure of the aortic root which leads to an increase in coronary flow. Balloon deflation which
Abstracts / Biomedicine & Pharmacotherapy 62 (2008) 503e512
takes place during late diastole achieves one of the main IABP therapeutic effects by reducing left ventricular afterload. Unavoidably, the balloon contacts the inner wall of the aorta with every inflation/deflation cycle. This repeated event and possible contact with atherosclerotic plaque have been reported to be responsible for balloon rupture. However, there has not been a methodical study to investigate the mechanical effects of balloon-wall interaction. For example, during inflation the balloon approaches the endothelium as it displaces a volume of blood proximally and distally. This squeezing process generates shear stresses, which hasn’t yet been quantified. Similarly, when the balloon moves away from the endothelium during deflation, it generates micro pressure differences that may impose stretching (pulling) stresses on the endothelium cells. Both of the above cases indicate that a very high spatial resolution is required in order to fully understand the process of interaction between device and endothelium, and to interpret the effects at the cellular level. E-mail address: [email protected] (M. Atherton).
5
isolated a novel cyclic depsipeptide, which we named heptadepsin, from Paenibacillus sp. As the mechanism of inhibition, we found that heptadepsin interacted directly with the lipid A moiety of LPS. We previously designed (-)-DHMEQ based on the structure of epoxyquinomicin C as a specific inhibitor of NF-kB. We reported that (-)-DHMEQ reduced adhesion molecule expressions and inhibited mononuclear cell or cancer cell-HUVEC adhesion especially in flow. We have recently elucidated the molecular target of (-)-DHMEQ, which is NF-kB itself. Thus, we have isolated from nature or synthesized several compounds that modify the functions of endothelial cells. These compounds may be useful for the mechanistic studies and also for the development of new anti-inflammatory or anticancer agents. E-mail address: [email protected]
6 The cerebrovascular endothelium: a target for neuroprotection Ferenc Bari 1, Ferenc Domoki
Screening of microbial secondary metabolites that inhibit endothelial cell growth Kazuo Umezawa Center for Chemical Biology, School of Fundamental Science and Technology, Faculty of Science and Technology, Keio University, Yokohama 223-0061, Japan Microbial and plant-derived bioactive metabolites are a treasury of organic compounds having various structures and biological activities. So we looked for bioactive metabolites that are useful for the regulation of endothelial cell functions employing various screening systems. We isolated sangivamycin, an unusual nucleoside, from Streptomyces as the compound that selectively inhibited the growth of human umbilical vein endothelial cells (HUVECs). It inhibited the growth and DNA synthesis selectively in HUVECs. Angiostatin, a potent angiogenesis inhibitor, binds to the subunit of mitochondria-type ATP synthase on the surface of HUVECs, and inhibits endothelial cell proliferation and migration. The F1 catalytic domain of extracellular ATP synthase was also reported to be mandatory for the endothelial cell proliferation. These reports suggest that inhibition of ATP synthase may selectively inhibit the growth of endothelial cells. We recently found that sangivamycin inhibited angiogenesis, at least in part, by suppression of ATP synthase activity on the endothelial cell surface. Recently, we also demonstrated the anti-angiogenic activity of sangivamycin in vivo. Sangivamycin inhibited the in vivo angiogenesis within chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays. We searched for compounds that could inhibit lipopolysaccharide (LPS)-stimulated adhesion between HUVECs and myelocytic cell line HL-60 cells. In the course of our screening we
505
1,2
, David W. Busija
2
1
Department of Physiology, Faculty of Medicine, University of Szeged, Hungary 2 Department of Physiology & Pharmacology, Wake Forest University Health Sciences, Winston-Salem, USA Cerebral ischemia/reperfusion (IR) causes endothelial dysfunction that may play a critical role in the pathogenesis of ischemic brain injury. Since the effective therapeutic interventions are very limited, there has been intensive search for innovative therapeutic strategies including the induction of endogenous protection and cellular repair of the blood vessels. Preconditioning (PC) is a phenomenon in which brief nonlethal episode(s) of ischemia, or the administration of certain pharmacological agents initiate mechanisms leading to protection against subsequent, prolonged periods of ischemia or other lethal stress. Pharmacological preconditioning of cerebral blood vessels may be an important strategy for reducing vascular dysfunction following IR. Recently, we have provided evidence that the mitochondrial ATP-sensitive potassium channel (mKATP) opener diazoxide (DIAZ) elicits preconditioning effects on the cerebrovascular endothelium in several in vivo models of cerebral ischemia (1,2). In rats, we investigated the effects of DIAZ on blood-brain barrier (BBB) function during IR injury. Rats were treated with 6, 20 or 40 mg/kg DIAZ ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan’s-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. IR increased BBB permeability for the large molecular weight EB (ng/ mg) in the cortex which was significantly attenuated in DIAZ-treated rats. DIAZ pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after diazoxide pretreatment. In anesthetized newborn piglets, we tested
504
shear stress, whereas susceptible regions, e.g. arches and branches, are exposed to low / oscillatory shear. Shear stress regulates vascular inflammation and atherosclerosis by altering the physiology of endothelial cells (EC), which detect shear stress via a tri-molecular complex expressed at their surface. High shear stress suppresses EC activation by inhibiting pro-inflammatory NF-kB and MAP kinase e AP-1 signaling via mechanisms that involve the activation of KLF2 and Nrf2 transcription factors. In contrast, low and oscillatory shear enhances endothelial activation via activation of MAP kinase e AP-1 and NF-kB pathways. These mechanisms are likely to contribute to the focal distribution of atherosclerotic lesions and could potentially be exploited to design novel therapeutic strategies to protect susceptible regions of the arterial tree from inflammation and atherosclerosis. E-mail address: [email protected]
3 Leukocyte-Endothelial Interactions with Respect to the Adhesion Cascade Dr Victoria Ridger Cardiovascular Research Unit, School of Medicine and Biomedical Sciences,University of Sheffield, UK Leukocyte recruitment to the site of injury and inflammation occurs via a sequence highly regulated events, collectively known as the adhesion cascade. They are characterised as capture, rolling, slow rolling, crawling, adhesion and extravasation. At each step, leukocytes interact with the endothelium through adhesion molecules; the selectins and their ligands regulate the first contact events and rolling whereas the integrins are involved in the later stages. Not only do these molecules control the physical interactions between leukocytes and endothelial cells, but they also are involved in signalling and preparing the leukocyte for the subsequent steps in the cascade. The onset of inflammation increases the expression and alters the avidity of these adhesion molecules on both the leukocytes and the endothelium, increasing the adhesive environment. Marginated leukocytes make initial contact with endothelial cells through interaction of L- (on leukocyte) or P- (on endothelial cells) selectins with their respective ligands. Rolling then occurs through a series of bond formations at the leading edge and breakages at the trailing edge of the leukocyte. The next stage of the cascade is slow rolling, mediated by E-selectin, observed after longer periods of endothelial stimulation. Slow rolling is thought to play a role in prolonging the exposure of rolling leukocytes to chemokines. Rolling also triggers cell signalling events thought to prepare the cell for the next step of ‘‘firm’’ adhesion. Chemokines are involved in the transition of rolling into firm adhesion and in the shedding of L-selectin from the surface of leukocytes. Firm adhesion is known to be highly dependent on b2 integrins and their ligands, members of the immunoglobulin superfamily. Once firmly adherent, leukocytes crawl towards endothelial-endothelial cell junctions via these same interactions. Transendothelial migration is
generally thought to occur in an intercellular manner and is mediated by integrin PECAM-1, ICAM-2, JAM-A and CD99. However this latter stage is not as well characterised. The understanding of the adhesion cascade and the multiple steps involved in leukocyte recruitment is essential for the development of effective, novel therapies for inflammatory diseases. E-mail address: [email protected]
4 Vascular device interaction with the endothelium Mark Atherton 1, Ashraf W Khir 1, Marco Cavazzuti 2, Giovanni Barozzi 2, & Michael Collins 1 1
School of Engineering and Design, Brunel University, West London, UK 2 Dipartminto di Ingegneria Meccanica e Civile, Universita` di Modena e Reggio Emilia, Italy Cerebral stents and Intra Aortic Balloon Pumps (IABP) are examples of mechanical devices that are inserted into arteries to restore flows to clinically healthy states. The stent and the IABP ‘correct’ the arterial flow by static dilation and by cyclical occlusion respectively. As this presentation shows, these functions are effectively modelled by current engineering practice. As interventions however, by their very nature they involve physical contact between a non-biological structure and the sensitive endothelial surface. The possible damage to the endothelium is not currently well addressed and we also consider this issue. Cerebral stents generally have two primary clinical objectives: to mechanically dilate a stenosed artery and to have minimal detrimental impact upon local blood flow characteristics. These objectives are well served at the arterial scale as these devices are evidently effective in opening up diseased arteries and restoring vital flows. However, at the near-wall micro-scale the picture is less satisfactory, as thin stent wires apply stresses to the endothelium and glycocalyx and the local flow is disturbed rather than being ideally streamlined. This causes further interaction with this endothelium topography. Wall Shear Stress (WSS) is the measure commonly used to indicate the interaction between fluid and wall but it is a broad brush approach that loses fidelity close to the wall. We will present simulation results of blood flow through a stented cerebral saccular aneurysm under these limitations of WSS. The Intra Aortic Balloon Pump (IABP) is a widely used temporary cardiac assist device. The balloon is usually inserted from the iliac artery, advanced in the aorta until it reaches the desired position; with its base just above the renal bifurcation and the tip approximately 10cm away from the aortic valve. The balloon is inflated and deflated every(1:1), every other- (1:2) or every second (1:3) cardiac cycle. Balloon inflation, which takes place during early diastole, causes an increase in the pressure of the aortic root which leads to an increase in coronary flow. Balloon deflation which
Abstracts / Biomedicine & Pharmacotherapy 62 (2008) 503e512
takes place during late diastole achieves one of the main IABP therapeutic effects by reducing left ventricular afterload. Unavoidably, the balloon contacts the inner wall of the aorta with every inflation/deflation cycle. This repeated event and possible contact with atherosclerotic plaque have been reported to be responsible for balloon rupture. However, there has not been a methodical study to investigate the mechanical effects of balloon-wall interaction. For example, during inflation the balloon approaches the endothelium as it displaces a volume of blood proximally and distally. This squeezing process generates shear stresses, which hasn’t yet been quantified. Similarly, when the balloon moves away from the endothelium during deflation, it generates micro pressure differences that may impose stretching (pulling) stresses on the endothelium cells. Both of the above cases indicate that a very high spatial resolution is required in order to fully understand the process of interaction between device and endothelium, and to interpret the effects at the cellular level. E-mail address: [email protected] (M. Atherton).
5
isolated a novel cyclic depsipeptide, which we named heptadepsin, from Paenibacillus sp. As the mechanism of inhibition, we found that heptadepsin interacted directly with the lipid A moiety of LPS. We previously designed (-)-DHMEQ based on the structure of epoxyquinomicin C as a specific inhibitor of NF-kB. We reported that (-)-DHMEQ reduced adhesion molecule expressions and inhibited mononuclear cell or cancer cell-HUVEC adhesion especially in flow. We have recently elucidated the molecular target of (-)-DHMEQ, which is NF-kB itself. Thus, we have isolated from nature or synthesized several compounds that modify the functions of endothelial cells. These compounds may be useful for the mechanistic studies and also for the development of new anti-inflammatory or anticancer agents. E-mail address: [email protected]
6 The cerebrovascular endothelium: a target for neuroprotection Ferenc Bari 1, Ferenc Domoki
Screening of microbial secondary metabolites that inhibit endothelial cell growth Kazuo Umezawa Center for Chemical Biology, School of Fundamental Science and Technology, Faculty of Science and Technology, Keio University, Yokohama 223-0061, Japan Microbial and plant-derived bioactive metabolites are a treasury of organic compounds having various structures and biological activities. So we looked for bioactive metabolites that are useful for the regulation of endothelial cell functions employing various screening systems. We isolated sangivamycin, an unusual nucleoside, from Streptomyces as the compound that selectively inhibited the growth of human umbilical vein endothelial cells (HUVECs). It inhibited the growth and DNA synthesis selectively in HUVECs. Angiostatin, a potent angiogenesis inhibitor, binds to the subunit of mitochondria-type ATP synthase on the surface of HUVECs, and inhibits endothelial cell proliferation and migration. The F1 catalytic domain of extracellular ATP synthase was also reported to be mandatory for the endothelial cell proliferation. These reports suggest that inhibition of ATP synthase may selectively inhibit the growth of endothelial cells. We recently found that sangivamycin inhibited angiogenesis, at least in part, by suppression of ATP synthase activity on the endothelial cell surface. Recently, we also demonstrated the anti-angiogenic activity of sangivamycin in vivo. Sangivamycin inhibited the in vivo angiogenesis within chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays. We searched for compounds that could inhibit lipopolysaccharide (LPS)-stimulated adhesion between HUVECs and myelocytic cell line HL-60 cells. In the course of our screening we
505
1,2
, David W. Busija
2
1
Department of Physiology, Faculty of Medicine, University of Szeged, Hungary 2 Department of Physiology & Pharmacology, Wake Forest University Health Sciences, Winston-Salem, USA Cerebral ischemia/reperfusion (IR) causes endothelial dysfunction that may play a critical role in the pathogenesis of ischemic brain injury. Since the effective therapeutic interventions are very limited, there has been intensive search for innovative therapeutic strategies including the induction of endogenous protection and cellular repair of the blood vessels. Preconditioning (PC) is a phenomenon in which brief nonlethal episode(s) of ischemia, or the administration of certain pharmacological agents initiate mechanisms leading to protection against subsequent, prolonged periods of ischemia or other lethal stress. Pharmacological preconditioning of cerebral blood vessels may be an important strategy for reducing vascular dysfunction following IR. Recently, we have provided evidence that the mitochondrial ATP-sensitive potassium channel (mKATP) opener diazoxide (DIAZ) elicits preconditioning effects on the cerebrovascular endothelium in several in vivo models of cerebral ischemia (1,2). In rats, we investigated the effects of DIAZ on blood-brain barrier (BBB) function during IR injury. Rats were treated with 6, 20 or 40 mg/kg DIAZ ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan’s-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. IR increased BBB permeability for the large molecular weight EB (ng/ mg) in the cortex which was significantly attenuated in DIAZ-treated rats. DIAZ pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after diazoxide pretreatment. In anesthetized newborn piglets, we tested