The multi‐functional serpin,protein C inhibitor: beyond thrombosis and hemostasis

Summary. Protein C inhibitor (PCI) is a member of the serine protease inhibitor (serpin) family. PCI was initially found to be an inhibitor of activated protein C, and later shown to be a potent inhibitor of blood coagulation and fibrinolysis such as that mediated by urokinase type‐plasminogen activator. Therefore, the protein came to be known as plasminogen activator inhibitor‐3. It also inhibits proteases involved in fertilization. PCI is broadly conserved, and is found in human, rhesus monkey, cow, rabbit, rat, mouse and chicken. The human PCI gene is located on chromosome 14q32.1 in a cluster of genes encoding related serpins. Sp1‐ and AP2‐binding sites in the 5′‐flanking region act as promoter and enhancer, respectively, for its expression in the liver. PCI mRNA is expressed in many organs in primates, but only in the reproductive organs in rodents. Recent studies using transgenic mice expressing the human gene have suggested that PCI is also involved in regulation of lung remodeling, tissue regeneration, vascular permeability, proteolysis in the kidney and tumor cell invasion. A protease inhibitor‐independent activity of PCI, the prevention of anti‐angiogenesis and metastasis of tumor cells, has also been observed. Thus, PCI is a unique multi‐functional serpin playing diverse roles in the thrombosis and hemostasis in multiple organs and tissues of a variety of species.     

Influence of thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 gene polymorphisms on tissue-type plasminogen activator-induced recanalization in ischemic stroke patients

OBJECTIVE: Endogenous resistance to tissue-type plasminogen activator (t-PA) might decrease the benefit of thrombolysis-induced recanalization. Thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are fibrinolysis inhibitors. TAFI removes residues from partially degraded fibrin that in turn eliminates plasminogen binding sites; PAI-1 directly inhibits the activity of t-PA. We aimed to study whether the presence of two common functional polymorphisms of the TAFI and PAI-1 genes influence rates of recanalization of the middle cerebral artery (MCA) among t-PA-treated stroke patients. METHODS AND RESULTS: TAFI and PAI-1 polymorphism determinations were performed by restriction fragment length polymorphism mapping and conventional sequencing in 139 patients with strokes involving the MCA and who received t-PA within 3 h. Recanalization was diagnosed by means of transcranial Doppler. No association was found between PAI-1 4 G/5 G polymorphism and recanalization rate. Dyslipidemia and TAFI Thr325Ile polymorphism were the main variables associated with recanalization resistance by the end of t-PA infusion: odds ratio (OR) 4.1 [95% confidence interval (95% CI) 1.6-10.8, P = 0.003] and OR 5.6 (95% CI 1.2-20, P = 0.031), respectively. The combination of the two polymorphisms doubled the risk of absence of recanalization: OR 11.1 (95% CI 1.4-89.8, P = 0.025). CONCLUSIONS: Polymorphic fibrinolysis inhibitor genes influence t-PA-induced recanalization resistance in ischemic stroke patients, especially when coexisting in the same patient. Efforts to individualize thrombolytic treatments are required.     

The hyperfibrinolytic state of mice with combined thrombin‐activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor‐1 gene deficiency is critically dependent on TAFI deficiency

Summary. Background: Mice with single gene deficiency of thrombin‐activatable fibrinolysis inhibitor (TAFI) or plasminogen activator inhibitor‐1 (PAI‐1) have an enhanced fibrinolytic capacity. Objectives: To unravel the function and relevance of both antifibrinolytic proteins through the generation and characterization of mice with combined TAFI and PAI‐1 gene deficiency. Results: Mating of TAFI knockout (KO) mice with PAI‐1 KO mice resulted in the production of TAFI/PAI‐1 double‐KO mice that were viable, were fertile, and developed normally. In a tail vein bleeding model, the bleeding time and hemoglobin content of the TAFI/PAI‐1 double‐KO mice did not deviate significantly from those of the single‐KO mice or of the wild‐type (WT) counterparts. Interestingly, in ex vivo rotational thromboelastometry measurements with whole blood samples, TAFI KO mice and TAFI/PAI‐1 double‐KO mice were more sensitive to fibrinolytic activation with tissue‐type plasminogen activator than WT or PAI‐1 KO mice. This enhanced fibrinolytic capacity was confirmed in vivo in a mouse thromboembolism model, as shown by decreased fibrin deposition in the lungs of TAFI KO mice and TAFI/PAI‐1 double‐KO mice as compared with WT or PAI‐1 KO mice.Conclusions: TAFI gene inactivation predominantly contributes to the increased fibrinolytic capacity of TAFI and PAI‐1 double‐gene‐deficient mice, as observed in some basic thrombosis models.     

TAFIa, PAI-1 and α2-antiplasmin: complementary roles in regulating lysis of thrombi and plasma clots

PAI-1 and alpha(2)-antiplasmin (alpha(2)AP) are the principal direct inhibitors of fibrinolytic proteases. Thrombin activatable fibrinolysis inhibitor (TAFI), a plasma procarboxypeptidase activated by thrombin-thrombomodulin to form TAFIa, also regulates fibrinolysis by modulating fibrin. In this study, the relative contributions of PAI-1, alpha(2)AP and TAFIa to inhibition of lysis were assessed. In platelet-poor plasma clots, alpha(2)AP, TAFIa and PAI-1 all inhibited lysis, as shown by the addition of neutralizing antibodies to alpha(2)AP and PAI-1 +/- CPI, a potato carboxypeptidase inhibitor. alpha(2)AP played the largest role in regulating plasma clot lysis, but neutralization of inhibitors in combinations was more effective in shortening lysis times, with a maximal effect when all three inhibitors were neutralized. In platelet-rich clots, a larger contribution of PAI-1 was evident. Tissue plasminogen activator induced lysis of model thrombi, made from whole blood, was approximately doubled on incorporation of CPI, illustrating a substantial contribution of TAFIa to inhibition of thrombus lysis. Similar increases in thrombus lysis were observed on inclusion of neutralizing antibodies to PAI-1 and alpha(2)AP, with alpha(2)AP playing the dominant role. Maximal thrombus lysis occurred upon neutralization of all three inhibitors. These observations suggest that, despite the differences in concentrations and activities of inhibitors, and the different modes of action, the roles of the three are complementary in both plasma clot lysis and thrombus lysis.     

2 型糖尿病微血管病变患者纤溶系统变化与胰岛素抵抗的关系

目的 探讨2型糖尿病微血管病变患者纤溶活性变化与胰岛素抵抗(IR)之间的关系.方法 采用酶联免疫吸附试验测定53例2型糖尿病惠者(包括无血管并发症组30例和微血管并发症组23例)和25例正常对照者血浆组织型纤溶酶原激活剂(t-PA)、纤溶酶原激活物抑制剂-1(PAI-1)含量,结合临床资料分析其变化趋势及影响因素.结果 2型糖尿病患者血浆t-PA含量明显降低,而PAI-1含量明显升高,t-PA分别是(7.09±2.10)μg/L vs(12.40±2.11)μg/L(P＜0.05),PAI-1分别是(40.38±1.74)μg/L vs(25.28±2.83)μg/L(P＜0.05),合并微血管病变者,此变化更为显著,t-PA分别(6.22±1.23)μg/L vs(12.40±2.11)/μg/L,PAI-1分别是(44.57±2.16)μg/L vs(25.28±2.83)μg/L(均P＜0.01).多元逐步回归分析显示,HOMA模型胰岛素抵抗指教(HOMA-IR)是PAI-1升高的独立危险因素.结论 2型糖尿病患者纤溶活性降低,IR在降低其纤溶活性,并发微血管病变中起了重要作用.     

血浆TAT、PIC、TM及t-PAIC联合检测诊断肝癌血栓的临床价值

目的 探究血浆凝血酶抗凝血酶复合物(T A T)、纤溶酶-α2纤溶酶抑制物复合物(PIC)、血栓调节蛋白(T M)及组织纤溶酶原激活物-抑制剂1复合物(t-PA IC)联合检测诊断肝癌血栓的临床价值.方法 选取2019年1月至2020年6月该院收治的142例肝癌患者作为研究对象,按照是否形成静脉血栓分为血栓组(35例)...     

Antibodies to tissue-type plasminogen activator (t-PA) in patients with inflammatory bowel disease: high prevalence, interactions with functional domains of t-PA and possible implications in thrombosis

BACKGROUND: Patients with inflammatory bowel disease (IBD) have an increased prevalence of thromboembolic events. The pathogenetic mechanisms of these events include reduced fibrinolysis, which may be caused by antibodies to tissue-type plasminogen activator (t-PA). OBJECTIVES: To evaluate anti-t-PA antibodies in patients with IBD, considering clinical, biochemical and functional characteristics. PATIENTS AND METHODS: We immunoenzymatically measured anti-t-PA antibodies in plasma from 97 consecutive IBD patients and 97 age- and sex-matched healthy controls. We also assessed the antibody interactions with different epitopes of t-PA, the antibody inhibition on t-PA activity and the correlations with clinical features and other serum antibodies. RESULTS: IBD patients had higher median anti-t-PA antibody levels (5.4 U mL(-1) vs. 4.0 U mL(-1); P < 0.0001): 18 patients were above the 95th percentile of the controls (OR 5.3; 95% CI 1.7-16.3; P < 0.003), and the six with a history of thrombosis tended to have high levels (6.9 U mL(-1)). Anti-t-PA antibody levels did not correlate with IBD type, activity, location or treatment, or with age, sex, acute-phase reactants or other antibodies. The anti-t-PA antibodies were frequently IgG1 and bound t-PA in fluid phase; they recognized the catalytic domain in 10 patients and the kringle-2 domain in six. The IgG fraction from the three patients with the highest anti-t-PA levels slightly reduced t-PA activity in vitro. CONCLUSIONS: The prevalence of anti-t-PA antibodies is high in IBD patients. By binding the catalytic or kringle-2 domains of t-PA, these antibodies could lead to hypofibrinolysis and contribute to the prothrombotic state of IBD.     

血栓分子标志物对下肢深静脉血栓形成的诊断及抗凝治疗效果的评价

目的观察下肢深静脉血栓患者血栓分子标志物的变化,分析其对静脉血栓形成诊断及疗效评估的价值。方法选取2018年12月至2019年5月北京积水潭医院创伤骨科下肢骨折术后深静脉血栓形成(DVT)患者64例为DVT组,未诊断为DVT患者39例为非DVT组;同期体检健康者28例为健康人对照组。采集静脉血并检测初诊时及利伐沙班抗凝治疗1个月后血栓分子标志物,即凝血酶-抗凝血酶复合物(TAT)、纤溶酶-α_2纤溶酶抑制剂复合物(PIC)、组织型纤溶酶原激活物-纤溶酶原激活物抑制剂复合物(t-PAIC)及可溶性血栓调节蛋白(sTM),并进行统计学分析。结果 DVT组血浆TAT、PIC、t-PAIC、sTM水平均高于非DVT组(P均0.05),DVT组血浆TAT、PIC、t-PAIC水平均高于健康人对照组(P均0.05),DVT组血浆sTM水平与健康人对照组差异无统计学意义(P0.05)。TAT、PIC、t-PAIC诊断DVT的ROC曲线下面积分别为0.997、0.980、0.758,约登指数最大时取cut-off值,cut-off值分别为2.45 ng/mL、0.715μg/mL、0.758 ng/mL;根据测定值cut-off值为阳性,测定值cut-off值为阴性,抗凝治疗1个月前后患者组TAT、PIC、t-PAIC水平及阳性率差异均有统计学意义(P均0.05)。结论血栓分子标志物TAT、PIC、t-PAIC能有效发现下肢DVT,同时对评估DVT患者抗凝疗效有意义。     

The effects of sampling from a peripheral venous catheter compared to repeated venepunctures on markers of coagulation,inflammation, and endothelial function

Abstract

Peripheral venous (PV) catheters are often used for serial blood sampling, but studies suggest that PV catheters increase markers of coagulation activation and inflammation. Whether the increase is caused by irritation of the vessel wall or diurnal variation is unknown. We therefore compared the effects of a PV catheter and repeated venepunctures on markers of coagulation, inflammation, and endothelial function.

A PV catheter was inserted at 07:45 in a hand vein in 10 healthy subjects, and blood samples were collected at 8:00, 10:00, 12:00, and 14:00. In the contralateral arm, blood was simultaneously obtained by venepunctures. Measures of coagulation, i.e., activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, prothrombin fragment 1?+?2 (F1?+?2) and thrombin-antithrombin (TAT), inflammation, i.e., interleukin 6 (IL-6) and C-reactive protein (CRP), and endothelial function, i.e., plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), von Willebrand factor (vWF), and tissue factor (TF) were measured in plasma.

The concentrations of TAT and F1?+?2 were significantly increased (10:00; p?<?.01, 12:00; p?<?.05, and 14:00; p?<?.01) in PV catheter samples compared with venepuncture samples. There was a minor increase in PT and INR and no increase in APTT, fibrinogen, CRP, PAI-1, tPA, vWF, and TF, with no differences between sampling methods. IL-6 concentrations increased in many PV catheter samples and venepuncture samples, but the response varied between the subjects.

Blood collection through a PV catheter induces coagulation activation, whereas endothelial function is not affected. More studies are needed to disclose the effect of blood sampling on IL-6.     

Renal synthesis of urokinase type-plasminogen activator, its receptor, and plasminogen activator inhibitor-1 in diabetic nephropathy in rats: modulation by angiotensin-converting-enzyme inhibitor

Plasmin is an important factor in the degradation of extracellular matrix. In the study reported here we examined the expression of plasminogen-activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (uPA), and uPA receptor (uPAR), as well as the relevance of such expression to the production of type IV collagen, a major component of extracellular matrix, in the renal tissue of rats with streptozotocin-induced diabetes. Because angiotensin II is involved in the synthesis of PAI-1 and uPA, we also examined the effect of benazepril, an angiotensin-converting-enzyme inhibitor, on the expression of PAI-1, uPA, and uPAR messenger RNAs (mRNAs) and type IV collagen protein. Rats with streptozocin-induced diabetes-some untreated and some treated with 30 mg/L benazepril-and nondiabetic control rats were sacrificed at 4, 12, or 24 weeks after induction of diabetes. We examined the expression of PAI-1, uPA, and uPAR mRNAs through the use of in situ hybridization and that of type IV collagen by means of immunohistochemical methods. In control rats, we detected weak signals for PAI-1, uPA, and uPAR mRNAs in glomeruli. Diabetic rats exhibited high levels of expression of PAI-1, uPA, and uPAR mRNAs and type IV collagen protein, mainly in mesangial cells. These mRNAs were synthesized in various renal cells (epithelial, mesangial, and endothelial cells and Bowman's capsule). Benazepril inhibited increases in all 3 mRNAs, especially in the mesangium; reduced type IV collagen expression; and attenuated mesangial expansion. Our results indicated that altered expression of PAI-1, uPA, and uPAR in diabetic nephropathy was associated with mesangial expansion and that the beneficial effects of ACE-I may be at least associated with such expression.     

Relationships among tumor burden, tumor size, and the changing concentrations of fibrin degradation products and fibrinolytic factors in the pleural effusions of rabbits with VX2 lung tumors

The VX2 tumor is derived from a papilloma virus-induced rabbit epithelial cell line. If VX2 tumor cells (trapped in a plasma clot) are introduced intravenously into NZW rabbits, the cells lodge in the lung capillary bed and produce tumors. Independently of the tumor burden (ie, the total tumor weight per rabbit), approximately 15% of rabbits with VX2 lung tumors accumulate an effusion in the interpleural space and this pleural effusion contains products of hemostasis. We hypothesized that these products were of intra-tumoral origin and that they changed in concentration as tumor burden increased. Interrelationships among lung-, tumor-weights, and pleural effusion volumes, and the concentrations of fibrinolytic factors, their catabolic products, and other proteins of pleural effusions were measured in rabbits with a wide range of tumor burdens. Positive correlations between tumor burden and total lung weight and between pleural effusion volume and net lung weight suggested that interstitial fluid from the stroma of tumors passed directly into the extravascular space of the lung(s) and into the interpleural space(s). Analyses of pleural effusions indicated that plasminogen-, alpha(2)-antiplasmin-, and plasminogen activator inhibitor-1-related proteins, urokinase-like- and tissue-plasminogen activator activities, and vascular endothelial growth factor increased in concentration up to a tumor burden of approximately 20-25 g. Plasmin activity and intact fibrinogen were absent. The concentration of fibrin(ogen) degradation products did not change significantly up to a tumor burden of approximately 25 g but increased substantially as tumor burdens exceeded 25 g. In conclusion, interstitial fluid from tumors enters the extravascular space of the host and may accumulate with fluid from non-tumor sources as a pleural effusion. The concentrations of fibrinolytic factors and their products in pleural effusions reflect the tumor burden of the rabbit. Conceivably, the components of a malignant effusion contain much information about the extent of tumor growth.     

Low molecular weight heparin (dalteparin) is equally effective as unfractionated heparin in reducing coagulation activity and perfusion abnormalities during the early treatment of pulmonary embolism

Little is known about the differences between unfractionated heparin (UFH) and low molecular weight heparin (LMWH) with regard to their effects on coagulation activity during treatment for pulmonary embolism. The objective of this study was to compare UFH and LMWH (dalteparin) in the early treatment of pulmonary embolism in terms of control of coagulation markers and perfusion abnormalities. Thirty-seven patients with acute pulmonary embolism were randomized to receive intravenous UFH or subcutaneous dalteparin, each accompanied by acenocoumarol. Daily blood samples were obtained for the measurement of thrombin generation (fragments 1 and 2 [F1+2], thrombin-antithrombin (TAT) complexes and fibrin monomers [FMs]) and fibrinolysis (d-dimer concentrations and clot-lysis times). Ventilation-perfusion scintigraphies were performed, and with the data they yielded, percentage of vascular obstruction scores (PVOs) were calculated on days 0 and 5. The international normalized ratio was within the therapeutic range in both groups on day 3. F1+2 and TAT complexes rapidly normalized, without differences between the groups (P =.5 and.4, respectively). FM levels did not decrease and, in fact, showed an increase in the UFH group from day 3 on (P <.05 between groups). d-Dimer levels decreased over time, with no differences between groups (P =.6). Clot-lysis times were shorter in the UFH group (P <.05). PVOs on days 0 and 5 were not different (P =.5 and.8, respectively), but the decrease in PVOs over time was greater in the dalteparin group (P =.04). These results show that dalteparin is at least as effective as UFH in reducing coagulation activity and perfusion abnormalities in the early treatment of pulmonary embolism.     

Fibrinogen catabolism within the procoagulant VX-2 tumor of rabbit lung in vivo: Effluxing fibrin(ogen) fragments contain antiangiogenic activity

Many types of solid tumors are known to be procoagulant environments. This is partly because a hyperpermeable vascular system within the tumor allows plasma hemostatic factors to accumulate in relatively high concentrations in the stroma, and many solid-tumor cells express tissue factor or a procoagulant factor. These circumstances appear to exist in the VX-2 lung tumor of the New Zealand White (NZW) rabbit, and they sustain a measurable turnover of stromal deposits of fibrin(ogen). We have measured the turnover of fibrinogen within tumors of the VX-2 tumor-burdened rabbit and analysed the catabolic products of fibrin(ogen) and the status of fibrinolysis in tumor-derived interpleural effusate. Using intravenously injected (125)I-labeled rabbit fibrinogen as a marker, we found that fibrinogen (approximate blood concentration 1740 microg/mL) passed from blood to VX-2 tumor stroma, saturating the tumor at a concentration of approximately 348 microg fibrinogen/g in approximately 12 hours. We measured fibrin(ogen) fragments, at a concentration of approximately 292 microg/mL, in interpleural effusates that we recovered from 13% of the VX-2-burdened rabbits. Unreduced fibrin(ogen) fragments consisted of 4 major components with a relative molecular mass of approximately 250,000 (assumed to be fragment X; approximately 9% of total fragments from densitometry of immunoblots), 200,000 (d-dimer; 41%), 110,000 (fragment D; 49%), and 50,000 to 55,000 (fragment E; 1%-2%) kD. Total fibrin(ogen) fragments immunopurified from effusates exhibited an antiangiogenic effect when subjected to a chick embryo chorioallantoic membrane procedure. Interpleural effusates were devoid of plasmin activity or active plasminogen activator inhibitor-1 but contained plasmin complexes and active urokinase-like plasminogen activator (uPA), alpha(2)-antiplasmin, and thrombin-activatable fibrinolysis inhibitor. We speculate that VX-2 cells release uPA to activate fibrinolysis within the tumor stroma. Catabolic products of hemostasis (eg, fibrinolytic fragments, angiostatin) flux from the stroma into the interpleural space, thereby providing a net antiangiogenic property to the effusate and ultimately to the lymphatic and circulatory systems.     

Idiopathic intracranial hypertension, polycystic-ovary syndrome, and thrombophilia

We studied thrombophilia, hypofibrinolysis, and polycystic-ovary syndrome (PCOS) in 65 women consecutively referred because of idiopathic intracranial hypertension (IIH) as a means of better understanding the origin of IIH, with the ultimate goal of developing novel medical therapies for IIH. Our hypothesis: IIH results in part from inadequate drainage of cerebrospinal fluid (CSF) resulting from thrombotic obstruction to CSF resorption-outflow, favored by thrombophilia-hypofibrinolysis. We conducted the polymerase chain reaction (PCR) and assessed serologic coagulation measures in 65 women (64 of them white) with IIH, PCR in 102 healthy white female controls (72 children, 30 age-matched adults), and serologic measures in the 30 adults. Of the 65 patients, 37 (57%) were found to have PCOS; 16 (43%) were obese (BMI > or = 30 to < 40), and 19 (51%) were extremely obese (BMI > or = 40). Of the 65 women with IIH, 25 (38%) were homozygous for the thrombophilic C677T MTHFR mutation, compared with 14% of controls (14/102) ( P = .0002). Thrombophilic high concentrations of factor VIII (>150%) were present in 9 of 65 (14%) IIH cases, compared with 0 of 30 controls (0%) (Fisher's p [p f ] = .053). An increased concentration of lipoprotein A (> or = 35 mg/dL), associated with hypofibrinolysis, was present in 19 of 65 IIH cases (29%), compared with 3 of 30 controls (10%) (p f = .039). IIH occurred in 18 of 65 IIH patients taking estrogen-progestin contraceptives (28%), in 6 patients taking hormone-replacement therapy (9%), and in 5 pregnant subjects (8%). We speculate that PCOS, associated with obesity and extreme obesity, is a treatable promoter of IIH. We also speculate that if thrombophilia-hypofibrinolysis and subsequent thrombosis are associated with reduced CSF resorption in the arachnoid villi of the brain, thrombophilia and hypofibrinolysis-often exacerbated by thrombophilic exogenous estrogens, pregnancy, or the paradoxical hyperestrogenemia of PCOS-are treatable promoters of IIH.     

Autonomic nerves and perivascular fat: Interactive mechanisms

The evidence describing the autonomic innervation of body fat is reviewed with a particular focus on the role of the sympathetic neurotransmitters. In compiling the evidence, a strong case emerges for the interaction between autonomic nerves and perivascular adipose tissue (PVAT). Adipocytes have been shown to express receptors for neurotransmitters released from nearby sympathetic varicosities such as adrenoceptors (ARs), purinoceptors and receptors for neuropeptide Y (NPY). Noradrenaline can modulate both lipolysis (via α₂- and β₃-ARs) and lipogenesis (via α₁- and β₃-ARs). ATP can inhibit lipolysis (via P₁ purinoceptors) or stimulate lipolysis (via P_2y purinoceptors). NPY, which can be produced by adipocytes and sympathetic nerves, inhibits lipolysis. Thus the sympathetic triad of transmitters can influence adipocyte free fatty acid (FFA) content. Substance P (SP) released from sensory nerves has also been shown to promote lipolysis. Therefore, we propose a mechanism whereby sympathetic neurotransmission can simultaneously activate smooth muscle cells in the tunica media to cause vasoconstriction and alter FFA content and release from adjacent adipocytes in PVAT. The released FFA can influence endothelial function. Adipocytes also release a range of vasoactive substances, both relaxing and contractile factors, including adiponectin and reactive oxygen species. The action of adipokines (such as adiponectin) and reactive oxygen species (ROS) on cells of the vascular adventitia and nerves has yet to be fully elucidated. We hypothesise a strong link between PVAT and autonomic fibres and suggest that this poorly understood relationship is extremely important for normal vascular function and warrants a detailed study.