Sites Of Vascular Lesions Research Articles

The function of ADAMTS13 in thrombogenesis in vivo: insights from mutant mice

Recently, two independent groups have established ADAMTS13-deficient mice using gene-targeting techniques. In humans, genetic or acquired deficiency in ADAMTS13 leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Surprisingly, ADAMTS13-deficient mice are viable with no apparent signs of TTP. However, these mouse models indicate that ADAMTS13 down-regulates platelet adhesion and aggregation in vivo, and ADAMTS13 deficiency can provide enhanced thrombus formation at the site of vascular lesions. In addition, ADAMTS13 by cleaving hyperactive ultra-large von Willebrand factor multimers not only down-regulates thrombosis but also inflammation. ADAMTS13-congenic mice that carry a truncated form of ADAMTS13 lacking the C-terminal domains have also been developed. Phenotypes of the congenic mice indicate the physiological significance of the C-terminal domains of ADAMTS13 in down-regulating thrombus growth. The studies mentioned here in different mouse models uncover the in vivo function of ADAMTS13 and strengthened the understanding of the mechanism of systemic disease TTP.

Impaired interaction between platelets and endothelial progenitor cells in diabetic patients

This editorial focuses on new data on the role of platelets of the recruitment of endothelial progenitor cells to sites of vascular lesion and their differentiation into endothelium. Questions of further research are raised.

Beyond Hemostasis: The Role of Platelets in Inflammation, Malignancy and Infection

Platelets play a complex role in hemostasis and thrombosis. The expression of multiple membrane receptors, both constitutive and activation-dependent, mediates platelet adhesion and aggregation at sites of vascular lesion. Platelet activation leads to exocytosis of granular constituents, release of newly synthesized mediators, and discharge of membrane-bound transcellular signaling molecules. Many of the same mechanisms that play a role in hemostasis and thrombosis facilitate platelet participation in other physiological or pathological processes including inflammation, malignancy and the immune response. Platelet receptors such as GPIb/IX/V, P-selectin, P-selectin glycoprotein ligand 1, CD40 and the alphaIIbbeta3 integrin, crucial to hemostasis, have been implicated in the progression of such inflammatory conditions as atherosclerosis, rheumatoid arthritis and inflammatory bowel disease, in the progression and metastatic spread of malignancies, and in the immune response to bacterial challenge. The release of platelet granular contents, including adhesive proteins, growth factors and chemokines/cytokines, that serve to facilitate hemostasis and wound repair, also function in acute and chronic inflammatory disease and in tumor cell activation and growth. Platelets contribute to host defence as they recognise bacteria, recruit traditional immune cells to the site of infection and secrete bactericidal mediators. The primary focus of this review is the "non-haemostatic" functions of platelets in physiological and pathological states.

The Evil in Atherosclerosis: Adherent Platelets Induce Foam Cell Formation

Platelet interaction with circulating progenitor cells plays an important role for repair mechanisms at sites of vascular lesions. Foam cell formation represents a key process in atherosclerotic plaque formation. We revealed that platelets regulate recruitment and differentiation of CD34 (+) progenitor cells into foam cells and endothelial cells. Adhesion studies showed that platelets recruit CD34 (+) progenitor cells via specific adhesion receptors, including P-selection/P-selectin glycoprotein ligand 1, and beta (1) and beta (2) integrins. CD34 (+) progenitor cells were coincubated with human platelets for 1 week. We demonstrated that a substantial number of CD34 (+) cells differentiated into foam cells. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and agonists of peroxisome proliferator-activated receptor-alpha and -gamma (PPAR-alpha and -gamma agonists) reduced this foam cell generation via inhibition of matrix metalloproteinase 9 secretions. Foam cell formation is also induced by low-density lipoproteins (LDLs). It was revealed that platelets take up modified LDL (fluorochrome-conjugated acetylated LDL) that is stored in the dense granules and internalized rapidly into the foam cells. These findings emphasize that the balance between endothelial cell regeneration and platelet-mediated foam cell generation derived from CD34 (+) progenitor cells may play a critical role in atherogenesis and atheroprogression.

Platelet-Induced Differentiation of Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) have the potential to home at sites of vascular lesions and to contribute to revascularization. This homing is a highly concerted mechanism, which involves chemotaxis, adhesion, migration, and finally integration of the cells into the target tissue. Only recently has the platelet been identified as a central mediator of EPC homing. Adherent platelets were able to mediate chemotaxis and adhesion of EPCs, a process that involved P-selectin glycoprotein ligand 1 and very late antigen-4 (VLA-4). Recent studies suggest that platelet-derived stromal cell-derived factor-1 is also involved centrally in the recruitment of EPCs. Furthermore, platelets induce progenitor cell migration by platelet-derived growth factor AB. Recent in vivo data confirm the recruitment of EPCs to sites of vascular lesions after vessel denudation by activated platelets and fibrin. Moreover, when coincubated with platelets, EPCs differentiate to mature endothelial cells and have the potential to migrate and colonize a platelet thrombus. The described interaction of EPCs with platelets represents a novel mechanism of vascular remodeling and healing of endothelial lesions.

Vascular dementia and the cholinergic pathways.

Vascular cognitive impairment/vascular dementia have been the subject of a large number of studies, due to their high prevalence and broad preventive and compensatory therapeutic potential. The knowledge of the cerebral anatomy correlated to the vascular territories of irrigation enables understanding of clinical manifestations, as well as classification into the several types of syndromic presentations. The central cholinergic system exercises important neuromodulatory functions on cerebral circuits related to cognitive and behavioral integration, as well as on vasomotor control related to cerebral blood flow adjustments. The acquisition of data on the anatomy of the cholinergic pathways, including the localization of the nuclei of the basal prosencephalon and the routes of their projections, established an important milestone. The knowledge of the vascular distribution and of the trajectories of the cholinergic pathways allows identification of the strategic points where a vascular lesion can cause interruption. The ensuing denervation leads to cholinergic hypofunction in the involved territories. This information proves important to better evaluate the sites of vascular lesions, emphasizing their strategic localizations in relation to the cholinergic pathways, and offering more robust foundations for treatment aiming at enhancing cholinergic activity.

Pharmacology of thienopyridines: rationale for dual pathway inhibition

Atherothrombotic disease is the result of atherosclerosis progression, and its clinical manifestations, mostly secondary to atherosclerotic plaque disruption and subsequent thrombus formation. At the site of vascular lesions, platelets adhere to the exposed matrix proteins, prompting platelet activation, resulting in the secretion of multiple platelet agonists, mostly modulated by intracellular calcium release. Among them, ADP, thromboxane A2, thrombin, and others play a critical role in maintaining a ‘pro-platelet-activating’ environment. Aspirin has been considered the gold standard of antithrombotic therapy. Its antiplatelet activity is achieved mainly by inhibiting arachidonic acid pathway (blocking thromboxane A2 release from platelets). ADP binds to platelet P2Y12 receptor, although this receptor is important in platelet aggregation induced by other agonists. Thienopyridines (ticlopidine and clopidogrel) exert their antiplatelet activity by binding to the P2Y12 receptor, irreversibly modifying it. Oral clopidogrel loading dose of 300–600 mg clopidogrel produces significant inhibition of ADP-induced platelet aggregation 2 hours after administration, which becomes maximal after 6 hours. Both aspirin and clopidogrel are ‘selective platelet-receptor’ inhibitors, and therefore are weak and safe antiplatelet agents. The co-administration aspirin-clopidogrel results in enhancement of platelet inhibition, since the act via different platelet receptors. Current research is focused in reversible or more potent ADP antagonists.

Adherent Platelets Recruit and Induce Differentiation of Murine Embryonic Endothelial Progenitor Cells to Mature Endothelial Cells In Vitro

The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti-P-selectin glycoprotein ligand-1 and anti-VLA-4 (beta1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.

Iloprost down-regulates the expression of the growth regulatory gene Cyr61 in human vascular smooth muscle cells

Prostacyclin and its mimetics have repeatedly been shown to act antiatherogenic and to inhibit neointima formation in several animal models of vascular injury. Treatment of human vascular smooth muscle cells with the prostacyclin mimetic iloprost (100 nm) drastically reduces expression of Cyr61, encoding the growth-regulatory cystein-rich angiogenic protein, without affecting the degradation rate of Cyr61 mRNA. Thrombin-induced Cyr61 expression was inhibited completely in the presence of iloprost. It is concluded that vasoprotective actions of prostacyclin in vivo may in part be due to inhibition of expression of the growth regulatory gene Cyr61 at sites of vascular lesions.

PPARalpha agonists inhibit tissue factor expression in human monocytes and macrophages.

Monocytic tissue factor (TF) expression may contribute to thrombogenicity associated with plaque rupture and may propagate thrombus formation at the site of vascular lesions. Induction of monocytic TF expression by endotoxin is mediated by the activation of transcription factors such as AP-1 and NF-kappaB. Both these signaling pathways are modulated by peroxisome proliferator-activated receptor-alpha (PPARalpha). Therefore, we have studied the effects of fibrates and other PPARalpha agonists on the expression of TF. We show that PPARalpha protein, like primary human monocytes, is also expressed in the human monocytic THP-1 cell line. Fenofibric acid, WY14643, and GW2331 inhibited TF mRNA upregulation after stimulation of THP-1 cells with lipopolysaccharide or interleukin-1ss. In primary human monocytes and macrophages, the lipopolysaccharide- or interleukin-1ss-mediated induction of TF activity was also inhibited by fenofibric acid, WY14643, or GW2331. These data indicate that activation of PPARalpha results in the downregulation of the TF gene. Our results suggest a novel role for PPARalpha in the control of atherosclerotic plaque thrombogenicity through its effects on TF expression in monocytes and macrophages.

Increased ICAM-1 and PECAM-1 Transcription Levels in the Heart of Apo-E Deficient Mice in Comparison to Wild Type (C57BL6).

Adhesion molecules and chemoattractants are thought to play a critical role in the homing of leukocytes to sites of vascular lesions. Apo-E deficiency in mice creates an atherosclerotic model that mimics vascular lesions in man. Little is known on the effect of Apo-E deficiency on expression of adhesion molecules in the hearts of these animals. In this study, male C57BL6 and Apo-E deficient mice were fed a chow diet over periods of time (0 to 20 weeks). The transcription levels of major adhesion molecules (ICAM-1, PECAM-1), present in the heart, were followed by northern blots. Immunohistochemistry was used to localize these adhesion molecules in the heart. Results show a significant increase in gene transcription levels of ICAM-1 and PECAM-1 in Apo-E animals, but not wild type, at 16 and 20 weeks of chow diet. Such increase in levels of transcription was not observed in younger Apo-E and C57BL6 animals (0, 6 weeks of diet). ICAM-1 and PECAM-1 were strongly expressed in the endocardium and heart microvessels. In contrast, VCAM-1 was poorly stained, with only an occasional expression on the endocardium and arterioles. Enhanced gene expression levels of heart ICAM-1 and PECAM-1 observed in Apo-E deficient mice, but not in control animals, appears to induce the initial stages of an inflammatory reaction. Such observations, not previously reported, may induce heart vascular remodeling.

Human endothelium: endovascular biopsy and molecular analysis.

To develop a safe and reproducible method for harvesting viable vascular endothelium to analyze gene expression at sites of vascular lesions. Coaxial curved stainless-steel guide wires were used to obtain samples of endothelial cells from large arteries and veins in 29 patients undergoing routine endovascular procedures. Three immunocytochemical markers were used to identify cells as endothelial. Cellular viability was evaluated in terms of cell membrane integrity, energy-dependent uptake of acetylated low-density lipoprotein, and cellular response to lipopolysaccharide. Single-cell reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study endothelial gene expression. Cells with endothelial morphology and immunoreactivity for von Willebrand factor, thrombomodulin, and angiotensin-converting enzyme were consistently obtained from iliac and carotid arteries and large veins (average yield [+/- standard error] from 26 iliac arteries, 262 endothelial cells +/- 45, 20%-30% of which were viable). These cells displayed induction of E-selection messenger RNA at PCR after exposure to lipopolysaccharide. Expression of vascular cell adhesion molecule 1 transcripts in endothelial cells increased with patient age (P < .01), whereas expression of intercellular adhesion molecule 1 did not. Viable endothelium can be obtained during routine angiography. Immunocytochemical and reverse transcription PCR analyses of these cells allowed determination of transcripts and proteins expressed by endothelium at sites of vascular lesions. Such information could aid in understanding mechanisms of vascular diseases and in clinical decision making.

Middle cerebral artery occlusive disease: The New England medical center stroke registry

To analyze the clinical features, vascular lesions, and infarct distribution in Asian and white patients with middle cerebral artery (MCA) territory ischemia, we studied age, sex, race, risk factors, angiographic, and neuroimaging findings among patients in the New England Medical Center Stroke Registry. We included patients with well-defined intrinsic occlusive lesions of the MCAs and patients with embolic MCA territory infarcts. Among 695 patients in the stroke registry, 89 (12.8%) qualified. They had 28 MCA intrinsic stenoses, 17 MCA embolic occlusions (cardiogenic or unknown origin), and 44 carotid artery (CA) stenoses or occlusions. MCA intrinsic disease patients were more often Asians and women, and more often had hypertension. Asians were older than whites. Coronary artery disease (27%), peripheral vascular disease (20.5%), and smoking (39%) were more common in CA disease patients. The most common site of MCA intrinsic stenosis (78%) and embolic occlusion (59%) was the mainstem MCA. Infarcts in patients with MCA intrinsic disease mostly involved the striatocapsular area (61%). Infarcts in patients with MCA embolic occlusion (75%) and CA disease (43%) most often involved the parietal lobe. In our hospital, most patients with MCA intrinsic disease are Asians and women and have hypertension and striatocapsular infarctions. Asian patients are usually older than white patients. The most common site of vascular lesions is the mainstem MCA.

Mutation and expression analysis of the endoglin gene in hereditary hemorrhagic telangiectasia reveals null alleles

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF-β binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant-negative model of receptor–complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice-site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT. Hum Mutat 11:286–294, 1998. © 1998 Wiley-Liss, Inc.

Mutation and expression analysis of the endoglin gene in hereditary hemorrhagic telangiectasia reveals null alleles.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF-beta binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant-negative model of receptor-complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice-site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT.

Crystal Structure of NMC-4 Fab anti-von Willebrand Factor A1 Domain

ABSTRACT: We have solved the crystal structure of the Fab fragment of NMC-4, a mouse monoclonal antibody that binds to the A1 domain of von Willebrand factor (vWF). Two Asp and three Tyr residues in the complementarity determining regions 1 and 3 of the heavy chain exhibited a spatial orientation suggestive of a dominant role in establishing contact with the antigen. A cluster of Asp and Tyr residues occurs also in a region of the platelet glycoprotein (GP) Ibα amino terminal domain known to be critically involved in vWF binding. Thus, the structural information obtained with NMC-4 may prove relevant to understand the stereochemical bases of the GP Ibα-vWF interaction essential for thrombus formation at sites of vascular lesion.

ドラッグデリバリーの空間的・時間的制御 薬物のターゲティング 基礎および臨床での証明

Prostaglandin E1 (PGE1), a vasodilator and platelet aggregation inhibitor, effectively treats many diseases. However, PGE1 is generated locally and acts locally to regulate physiological responses, thereby suppressing the progress of disease. Therefore, the therapeutic use of PGE1 necessitates the targeting of specific sites to maximize local action and prevent systemic side effects. The authors have developed a PGE1 preparation, using lipid microspheres (LM) with a 0.2 μ diameter (o/w emulsion) as carriers for passive targeting of sites of vascular lesions ; the preparation is called lipo-PGE1. Favorable preclinical and clinical results have been achieved with preparation. The intravascular distribution of lipo-PGE1 injected intravenously in spontaneously hypertensive rats(SHR) was investigated using electron microscopy. LM were accumulated in the subendothelial space of vascular walls, particularly in vascular lesions associated with hypertension. Laser confocal imaging revealed the marked uptake of PGE1 by endothelial cells of diseased or newly developed vessels treated with lipo-PGE1. On the other hand, free PGE1 showed trace accumulation in the endothelial cells. The control cells incubated with normal mouse IgG instead of anti-PGE1 antibody did not show any fluorescence. Lipo-PGE1 is delivered preferentially to the site of vascular lesions, and PGE1 becomes less irritant because it is incorporated into the microspheres. In addition, its inactivation in the lungs is slightly reduced for the same reason. All these features of the preparation strongly suggest that it would be very valuable in clinical practices. We performed the Phase III clinical trial of lipo-PGE1 conducted in Japan which clearly showed that lipo-PGE1 is safe and effective in patients with arteriosclerotic vascular disease, diabetic neuropathy, peripheral vascular disease secondary to diffuse connective tissue diseases, vibration disease, or ductus arteriosusdependent congenital heart disease.

Evaluation of the Basilar Artery Flow Velocity by TCD Examination

We measured flow velocities of the basilar artery (BA) via the suboccipital window by TCD in 115 subjects to investigate BA hemodynamics. Twenty one of the subjects had posterior circulation pathology, 37 had anterior circulation pathology, and the remaining 57 cases had no lesions. The vascular lesion was confirmed by cerebral angiography in all cases. The characteristic findings of the BA flow velocity by the site of vascular lesion were as follows. 1) Normal: signal obtainable from a depth of 80 mm to depths beyond 95 mm. Timeaveraged mean velocity was between 20 and 45 cm/s and velocity variations occurred at different depths<16 cm/s. 2) BA occlusion: a) Total occlusion: no signal or signal only from the site adjacent to the vertebrobasilar junction. b) Distal occlusion: continuous low velocity (<20 cm/s) .c) Proximal occlusion: continuous retrograde flow. 3) Stenosis of the BA: sudden increase in flow velocity at the stenotic portion or continuous low velocity proximal to the site of severe stenosis. 4) Occlusion of the bilateral vertebral arteries (VA) : flow from the anterior spinal artery detectable. 5) Unilateral occlusion of the VA: no distinctive flow pattern. 6) Internal carotid artery occlusion or vascular rich tumor in the posterior lobe: increased flow velocity along the BA. 7) Other anterior circulation pathology: no characteristic signal.

Role of platelet membrane glycoproteins and von Willebrand factor in adhesion of platelets to subendothelium and collagen.

Development of in vitro perfusion systems and quantitative methods to evaluate various aspects of platelet-surface interactions have helped to elucidate some complex interactions in platelet adhesion and platelet-platelet cohesion. The importance of platelet GPs in these interactions was soon evident, and rapid progress in biochemistry and immunology has made it possible to study the roles of these GPs in more detail. From our studies, which made use of platelets with GP deficiencies, MAbs, purified vWF, proteolytic fragments of vWF, a synthetic peptide, and in vitro perfusion systems, it appeared that GPIb and GPIIb/IIIa function as receptors for vWF, which binds platelets to connective tissue at sites of vascular lesions (FIGURE 8). More detailed information about these interactions is expected in the near future when bioengineered products are available for testing in flow models and in other experimental approaches.