Clinical Hemorheology and Microcirculation

The main function of the microvasculature is the controlled exchange of materials with surrounding tissues. This necessitates a large vessel surface established by a high number of vessels with small diameters and thus an inherently high individual resistance to flow. The hydrodynamic resistance of a microvascular network with given angioarchitecture depends on the apparent viscosity of blood flowing in the microvessels. Apparent viscosity declines with decreasing diameter (the Fahraeus–Lindqvist effect) and is minimal at diameters of about 5–7 μm due to the optimal alignment of red cells with the flow. In vivo, a number of additional phenomena influence blood rheology and network hemodynamics. The distribution of blood flow and red cell flux within networks is influenced by the mechanics of red cell motion at individual diverging bifurcations (phase‐separation effect). Furthermore, recent studies have revealed the presence of a thick endothelial surface layer (∼0.5 μm) on the luminal surface of microvessels which is attached to the endothelial glycocalyx. This layer modulates flow resistance and may be relevant for a number of other processes such as inflammatory responses and blood coagulation. Information on microvascular rheology can be used to develop mathematical models of network hemodynamics and vascular adaptation to the local environment (angioadaptation), to investigate the complex interrelated mechanisms which establish and maintain functionally adequate microvascular networks.

Pharmacological induction of angiogenesis is a new treatment of cerebrovascular insufficiency without surgical treatment. It is an urgent task to investigate the dynamic process of angiogenesis and of the microvascular perfusion of the cerebral neoplastic tissue in vivo. The present study is concerned with microcirculatory aspects of cerebral neocapillaries in vivo. A novel model of cerebral angiogenesis was developed by inducing cerebral neocapillaries in mice using growth factors such as basic fibroblast growth factor (bFGF) and platelet‐derived growth factor (PDGF). By a direct observation of the neocapillary microcirculation under a fluorescence videomicroscope, the neocapillary density, diameter and red cell velocity were measured to evaluate the development and remodeling of the neocapillaries with the number of days after incubation. The neocapillary response to topically applied acetylcholine (ACh) was examined by measuring changes in the diameter and red cell velocity. It was shown that PDGF‐induced neocapillaries was dilated in response to ACh on day 28 after incubation, while bFGF‐induced neocapillaries was not. Furthermore, the neocapillary pericytes were observed using confocal laser microscopy, based on the fluorescence immunohistochemical images of the neoplastic tissue. Several pericytes, stained with anti‐NG₂, appeared in the neocapillaries. It was suggested that these pericytes might be recruited in the neocapillaries to regulate blood flow without vascular smooth muscle.

Microcirculation conduit, distribution, exchange and reception vessels usually retain a demand‐dependent vascular–tissue match as well as a nutrient friendly capillary–matrix tissue match. Various stimuli can initiate a vascular–capillary matrix‐tissue mismatch. Counter‐regulatory mechanisms result in hyperplasia or apoptosis. Microvascular disease (MVD) as a consequence or outcome of supply–demand mismatch has clinical therapeutic and prognostic implications in the hypertensive syndrome and coronary artery disease (CAD) cases. Recognition of the role of apoptosis and MVD may initiate a paradigm shift in clinical practice. Digitalis and other anti‐hypertensive agents have anti‐apoptotic action and MVD blunting effects that can control LVH development to reduce congestive heart failure (CHF) progression.

Hepatic microvasculature receives blood from two types of afferent vessels: the terminal portal venule (TPVn) and the terminal hepatic arteriole (THAo). The TPVns directly connect with the capillary bed in the liver parenchyma, which is referred to as sinusoids. Hepatic arterial blood pours into the hepatic sinusoids not only indirectly via the anastomosis between the THAo and the portal venule (PVn), but also directly through the THAo or the capillaries derived from the arterial capillary network around the bile duct. From a regulatory point of view, the hepatic arterial system is considered to be supplementary, but hepatic arterial flow is essential for supplying oxygen to sinusoidal blood flow as well as to the bile ducts, portal venules and nerves in the portal tract. The main regulators of hepatic sinusoidal blood flow are present in the portal venous system. By intravital and scanning electron microscopy, it is evident that a potent vasoconstrictor endothelin (ET)‐1 causes a contraction of the SEF via the ET_B receptors, as well as a significant contraction of the PVn and TPVn, resulting in an increase in sinusoidal and pre‐sinusoidal microvascular resistance. This phenomenon implies that the TPVn, particularly the transitional part to the sinusoid, would provide an essential regulatory site for hepatic sinusoidal blood flow as an inlet sphincter‐like function. The endothelial cell linings along the hepatic sinusoids are characterized by the presence of a large number of sieve plate‐like pores, 100 nm in diameter, i.e. the sinusoidal endothelial fenestrae (SEF). The SEF are dynamic structures, forming the racemose invaginations of the endothelial plasma membrane across the endothelium, and regulating not only the permeability of hepatic sinusoids, but also the sinusoidal blood flow by the Ca⁺⁺‐actomyosin‐mediated contraction and dilatation of the SEF. Our recent immunoelectron microscopic and Western blot studies have revealed that caveolin‐1, i.e. the principal structural protein of caveolae, and endothelial nitric oxide synthase (eNOS) co‐exist in the plasma membrane of the SEF, implying that the SEF may correspond to a permanent (stationary) type of fused and interconnected caveolae, thus contributing to the local control of hepatic sinusoidal blood flow by the regulation of NO synthesis.

Glomerular endothelial cell (GEC) dysfunction due to oxidative stress and enhanced proinflammatory cytokines plays an important role in inducing proteinuria and procoagulant activity, namely blood hypercoagulability, hyperviscosity and local intravascular coagulation and altered hemorheology in NS. A dysfunctioning GEC releases fewer endothelium‐dependent vasodilators but produces more vasoconstrictors. Severe intrarenal hemodynamic alteration associated with hemodynamic maladjustment with preferential constriction at the efferent arteriole has been uniquely implicated in severe GN and NS‐FSGS. Such a constriction exerts three significant hemodynamic impacts. Proximal to the efferent arteriolar constriction, it induces (i) an overestimated GFR due to hyperfiltration and (ii) an elevated intraglomerular hydrostatic pressure. Distal to the efferent arteriolar constriction, it (iii) exaggeratedly reduces PTCF which correlates with the TIF.

A new technique is sweeping the world, and changing the course of human work and life. It is impacting upon models, methods and the development of medical research. In the development of this new technique, a huge quantity of experimental research and clinical practice has proved that many human diseases have a close relationship to pathological changes that take place in the microvascular system. It has been proven that the microvascular system is the target for studying disease development and the treatment of disease. Many studies have shown that successful pathogenesis and pathological research must be aimed at understanding the key proteins in cells, organs and systems, as well as investigating their interaction, and finding out how these proteins change under disease conditions. This paper reviews the current status of microvascular medicine and proteomics.

Asian traditional medicine (ATM) (herbal medicine, acupuncture or moxibution) has gained some popularity among communities in Asia, but there are still few evidences to prove the effectiveness of such therapeutic measures. A symposium was held with aim at the effectiveness of Asian traditional therapies in views of in vivo microcirculation. This report is concerned with the symposium, including Asian activities for ATM.

Angiogenesis offers an enormous potentials for therapeutic intervention of many disorders afflicting mankind at present. With the identification of the major molecular players involved in the sequence of events leading to the formation of new blood vessels from pre‐existing capillaries, inhibition or induction of the process may now be regulated. Bioactive compounds from natural sources may be used as regulatory agents. Inhibition of angiogenesis can control diseases characterized with excessive blood vessel growth like cancer, arthritis, psoriasis and diabetes retinopathy. Stimulation of angiogenesis would be favorable in the treatment of ischemic disorders and tissue engineering. An increasing number of bioactive compounds from natural sources and whose chemical structures have already been elucidated are reported as either potential inhibitors or inducers of angiogenesis. Drug development from natural products is a fast‐emerging field that needs to be supported to provide people with more readily available and affordable healthcare.

Glomerular endothelial dysfunction is believed to be responsible for the proteinuria and nephronal damage, namely tubulointerstitial fibrosis and glomerulosclerosis, observed in severe nephrosis such as focal segmental glomerulosclerosis. A dysfunctioning glomerular endothelium is likely to be induced by oxidative stress and oxidized LDL as well as altered immunocirculatory balance with a defective anti‐inflammatory pathway. A defective release of vasodilator inconjunction with enhanced production of angiotensin II induces hemodynamic maladjustment by preferential constriction at the efferent arteriole. Such a hemodynamic maladjustment exerts two significant hemodynamic impacts. Close to the efferent constriction, it induces intraglomerular hypertension and glomerulosclerosis. Far from the efferent constriction, it reduces peritubular capillary flow, which eventually leads to tubulointerstitial fibrosis. Treatment with a vasodilator improves the hemodynamic maladjustment but does not completely suppress proteinuria. A successful suppression of proteinuria is accomplished by using Ganoderma lucidum and vitamins C and E. The beneficial effect of Ganoderma lucidum appears to be multifactorial, including the modulation of immunocirculatory balance, antilipid, vasodilator, antiplatelet and improved hemorheology. Together with vitamins C and E, this helps to neutralize oxidative stress and suppress the toxic effect to the glomerular endothelial function.

Polydatin is extracted from a traditional Chinese herbal medicine, Polygonum cuspidatum, and has a special effect in shock treatment. The aim of this study is to explain the cellular and molecular basis of Polydatin in shock treatment. The fluorescent probe techniques, patch clamp method, and cellular flow chamber were used to test intracellular variables of vascular smooth muscle cells (VSMC), myocardial cells (MC), endothelial cells (EC), and white blood cell (WBC). It was shown that Polydatin could inhibit ICAM‐1 expression in EC stimulated by lipopolysaccharide (LPS), attenuate WBC‐EC adhesion, increase [Ca $^{2+}]\tsub{i}$ in MC with enhancement of MC contraction extent, activate K_ATP channels of VSMC, and decrease pH_i value and [Ca $^{2+}]\tsub{i}$ of VSMC in shock. The study suggests that Polydatin has multiple effects on VSMC, MC, WBC and EC, which are related to the enhancement of heart function and improvement of microcirculatory perfusion in shock.

Carica papaya seed extract is currently being marketed as a nutritional supplement with purported ability “to rejuvenate the body condition and to increase energy”. The product claims to improve immunity against common infection and body functioning. The present study was initiated to analyze the chemical constituents of the Carica^® Seed Extract and determine the potential immunomodulatory properties of the different bioactive fractions. These immunomodulatory activities of crude Carica^® Seed Extract and its bioactive fractions were examined in vitro using lymphocyte proliferation assays and complement‐mediated hemolytic assay. Three major observations were made in this study: (1) the crude Carica^® Seed Extract and two other bioactive fractions significantly enhanced the phytohemagglutinin responsiveness of lymphocytes; (2) none of the Carica^® Seed Extract (at the concentrations used in this study) was able to protect the lymphocytes from the toxic effects of chromium; and (3) some of the bioactive fractions of Carica^® Seed Extract were able to significantly inhibit the classical complement‐mediated hemolytic pathway. These findings provide evidence for immunostimulatory and anti‐inflammatory actions of Carica^® Seed Extract. No single compound is likely responsible for these activities. Further purification, isolation and characterization of the active components are needed.

This study was aimed at investigating the effects of extract of Ginkgo biloba (EGb) on cerebral vasospasm and microcirculatory perfusion after subarachnoid hemorrhage (SAH). An endovascular piercing method was used to induce Wistar rat SAH models, and animals were divided into sham‐operated, vehicle controls, and EGb‐treated groups. EGb was injected intraperitoneally 30 minutes before operation and was repeated every 6 hours, with a single dose of 15 mg/kg bw. Diameters of basilar arteries before and after operation were measured. Microcirculatory blood perfusion of parietal lobe cortex was detected using a laser Doppler flow‐meter probe within 24 hours. Endothelin‐1 levels in both plasma and brain tissue were detected at different time points. The results showed that SAH caused an immediate drop in microcirculatory blood flow in vehicle controls, which persisted for 24 hours. Endothelin‐1 levels in both plasma and brain tissue increased after SAH. EGb partly reversed spasms of the basilar artery and antagonized a drop in microcirculatory blood flow. EGb also prevented an increase in endothelin‐1 both in plasma and in brain tissue. In conclusion, EGb, by antagonizing the overproduction of endo‐ thelin‐1, partly reverses cerebral vasospasm and improves microcirculation, and thus relieves secondary ischemic brain injury after experimental SAH.

The effects of Aloe vera on microcirculation and levels of TNF‐α and IL‐6 were investigated in rats after inducing burn. Seventy‐two male Wistar Furth rats were equally divided into four groups as follow: controls (CON), untreated burn‐wound rats (BURN), normal saline‐treated burn‐wound rats (BURN‐NSS) and Aloe vera‐treated burn‐wound rats (BURN‐ALOE). The animals in each group were equally subdivided into three subgroups for the study on day 3, 7 and 14 post‐burn. Dorsal skinfold chamber preparation and intravital fluorescence microscopic technique were performed to examine leukocyte adhesion on postcapillary venules. ELISA techniques were performed to examine serum TNF‐α and IL‐6 levels. It was found that the amount of leukocyte adhesion was significantly reduced in the BURN‐ALOE group compared to rats in the BURN group on day 14. Levels of TNF‐α and IL‐6 were also decreased significantly compared to BURN at all three monitored time points. Aloe vera could inhibit the inflammatory process following burn injury, as characterized by the reduction of leukocyte adhesion, as well as those pro‐inflammatory cytokines.

Ipomoea muricata (L.) Jacq, locally known as ‘Tonkin’, has been used for generations by the Dominicans in the Philippines for medicinal purposes. The seeds, stems and leaves are said to be effective in treating several types of skin ailments such as chronic and gangrenous wounds, cuts and blisters due to burns. Scientific investigations to rationalize the reported medicinal uses of the plant were carried out at University of Santo Tomas. Botanists at the University did the identification and studied the cultivation and propagation of the plant. Pharmacists and chemists worked closely together on the ‘Tonkin’ seeds. The seeds were found to have both analgesic and antiseptic properties. Chemists identified indolizidine alkaloids in the seeds, to which the analgesic properties have been attributed. Antimicrobial and antifungal compounds were also identified. Different formulations of the crude drug have been made, namely, an ointment for the treatment of skin ailments, glycerol preparation for the treatment of pharyngitis and an otic preparation for the treatment of otitis externa. Limited clinical trials of these formulations were carried out in collaboration with the medical doctors. The clinical trial studies were supported by in vitro studies carried out by microbiologists. Basic research on this plant material is still continuing. Efforts are now underway for the commercial production of the Tonkin ointment.

The gastroprotective effect of quantified tannins (13.4%) from Syzygium cumini was determined. Gastric mucosal damage was induced in sixty eight rats by oral gavage administration of HCl/ethanol solution. For macroscopic and microscopic studies, 30 rats were divided into three groups consisting of a negative control, an Omeprazole group and a Tannins group. There was no significant difference in the number, size and surface area of macroscopic lesions between the three groups. Microscopic examination using Best's Ulcer Staging Index showed that Tannins had a very significant decrease in gastric mucosal damage with p<0.01. Average lymphocyte populations in the three groups showed no significant difference, although both the Tannins and Omeprazole group had fewer lymphocytes. Thirty‐eight rats were studied for the amount of free radicals present after induction of gastric damage. A dose which consisted of 20.0 g tannins/kg rat weight showed significantly lower stomach free radical concentrations. These findings suggest that tannins extracted from S. cumini have gastroprotective and anti‐ulcerogenic effects.

The oxygen binding capacity of human erythrocytes in selected solutions was studied in vitro. Three treatment groups were used in the study: 0.08% ι‐carrageenan solution, dextran 70, and normal saline solution (NSS). The different treatment groups were oxygenated. The percent oxygen saturation and oxygen partial pressure were determined by blood gas analysis, and oxyhemoglobin absorbance was measured using spectrophotometry. Analysis of variance (ANOVA) and Tukey's HSD Test were used to analyze the data. There were significant differences (p<0.05) observed in the percent oxygen saturations and oxygen partial pressures among the three treatment groups, with NSS having the highest values (97.65±0.385% and 160.18±152.567 mmHg, respectively) followed by dextran 70 (95.38±1.093% and 142.68±73.967 mmHg, respectively) then ι‐carrageenan (89.42±4.327% and 94.02±17.912 mmHg, respectively). However, there were no significant differences observed in the absorbance values of oxyhemoglobin among the three set‐ups. It was concluded that the oxygen binding capacity of erythrocytes in ι‐carrageenan solution is significantly lower than that in dextran 70 and NSS.

The objective of this study is to examine the effects of genistein on endothelial dysfunction in bilateral ovariectomized rats. Female Wistar rats were subjected to a bilateral ovariectomy (OVX rat). The animals were divided into three groups: sham treated with vehicle (DMSO 100 μl/day, Sham_veh), OVX treated with vehicle (DMSO 100 μl/day, OVX_veh), and OVX treated with genistein (0.25 mg/kg BW/day, OVX_gen). Mean arterial pressure (MAP), heart rate (HR), body weight (BW), uterine weight and plasma E₂ were monitored at 4‐week after the treatment. We investigated the endothelium‐dependent and ‐independent vasorelaxation by using acetylcholine (Ach 10⁻⁶ M) and sodium nitroprusside (SNP 10⁻⁷ M), respectively. The experimental results indicated that the uterine weights of all OVX rats were significantly decreased as compared to the sham groups (OVX $\tsub{veh}=0.007\pm 0.004$ g, OVX $\tsub{gen}=0.003\pm 0.001$ g, Sham $\tsub{veh}=0.017\pm 0.001$ g). MAP of OVX_veh group was significantly increased compared to the Sham group (OVX $\tsub{veh}=139.99\pm 7.50$ mmHg, Sham $\tsub{veh}=118.10\pm19.33$ mmHg, p<0.05). No significant increase in MAP was observed in OVX_gen (OVX $\tsub{gen}=123.33\pm 8.61$ mmHg; p<0.05). HR showed no significant difference among those groups. The present study of vasodilator responses demonstrated only the significant decrease in endothelium‐dependent, not for endothelium‐independence, in OVX rats, while the treatment of genistein could significantly attenuate this abnormality (OVX $\tsub{veh}=3.03\pm3.99$ %, Sham $\tsub{veh}=45.46\pm 3.59$ %, OVX $\tsub{gen}=33.52\pm 3.25$ % in % change of vessel diameter). The present findings suggest that genistein could be used as a therapeutic agent for menopausal vascular complications.

The effect of carrageenan against ischemia‐reperfusion injury was investigated. Rats were grouped into four, subjected to 10 min of ischemia and reperfusion in their mesenteric venules and 30% of their blood volume was replaced with either normal saline solution, dextran 70 in 5% dextrose, or iota‐carrageenan solution, while the fourth group remained untreated. Parameters such as venule diameter, red blood cell velocity, leukocyte rolling and adhesion, were then observed by video. Findings reveal that carrageenan infusion has the tendency to attenuate leukocyte rolling and adhesion, increase venule diameter and regain erythrocyte velocity after ischemia. The data suggest that the effects of carrageenan were comparable to that of either of the more commercially used plasma expanders, dextran 70 or normal saline solution. Overall, however, these did not differ significantly from the untreated rats.

We studied the early pathophysiological response of lenticulostriate arterioles in rats in three models of human conditions associated with stroke: (a) chronic angiotensin II‐hypertension; (b) chronic nicotine administration; (c) oxidative endothelial injury. In all three models, quantitative patch clamp analysis of freshly isolated vascular smooth muscle cells from lenticulostriate arterioles and posterior cerebral arteries showed significant increases in activity of functional L‐type calcium channels that were due to an increase in open channel probability, with no change in other biophysical properties or in channel expression. In addition, all three models showed evidence of endothelial dysfunction, but of a different nature in the three. With chronic angiotensin II‐hypertension, but not in the other two models, endothelial nitric oxide synthase (eNOS) was dysfunctional, was mislocalized away from its normal abluminal location, and was accumulated in peri‐nuclear Golgi. By contrast, the other two models showed no mislocalization of eNOS, but instead showed evidence of oxidative stress in endothelium, with up‐regulation of superoxide dismutase and hexose kinase. All three models showed significant up‐regulation of expression of proliferative cell nuclear antigen (PCNA) (PCNA index, 70–80%) in arterioles in situ, which is associated with increased activation of the nuclear transcription factor, phospho‐cAMP response element binding protein (phospho‐CREB). In addition, calmodulin‐dependent protein (CaM) kinase II was activated, in concert with the activation of L‐type calcium channels. Furthermore, blockers of either L‐type calcium channels (amlodipine) or of CaM kinase II (KN‐93) completely prevented the activation of CREB and the up‐regulation of PCNA in arterioles. Our findings demonstrate that abnormal regulation of L‐type calcium channels is directly responsible for abnormal proliferative responses in vascular smooth muscle in various forms of cerebral arteriolar injury associated with endothelial dysfunction.

Hypertension is not an isolated problem. Co‐morbidities of smoking, obesity, diabetes and dyslipidemia are all associated with microvascular disease (MVD) with abnormal PET scan and endothelial dysfunction. MVD may contribute to left ventricular hypertrophy (LVH) via an imbalance between hyperplasia and apoptotic signals. Digitalis and other anti‐hypertensive agents have anti‐apoptotic action and MVD blunting effects, respectively. Heart failure progression must then be based on the preservation of myocyte integrity. Indeed, altered contractility appears to be a consequence of rather than the cause of myocyte deterioration. LV systolic dysfunction improvement is already a late strategy. Furthermore, the efficacy of anti‐hypertension therapy may be limited in restoring LV diastolic function. Recognition of the role of apoptosis and MVD may initiate a paradigm shift in clinical practice.

A possible defensive mechanism in the basal region of the gastric mucosa was hypothesized in the present study. In vivo microscopy was performed to observe the basal region after thermal injury to the back skin of rats. A donor of nitric oxide, 3‐morpholinosydnonimine hydrochloride (SIN‐1), or a serine protease inhibitor, camostat mesilate, was administered. Anti‐vascular endothelial growth factor (VEGF) neutralizing antibody was administered 5 hours after thermal injury (anti‐VEGF group). Post‐capillary venules could be observed in the basal region of the gastric mucosa (PV‐BGM). The PV‐BGM was dilated 5 hours after thermal injury, and it was reduced by the administration of SIN‐1 or pre‐treatment with camostat mesilate. In the control group, the erosions did not reach the basal region of the gastric mucosa. Most of the erosions healed within 72 hours. Delayed healing was observed in the anti‐VEGF group. In this group, exudation and congestion in the basal region were observed at 24 hours, and ulcer formation was observed at 72 hours after thermal injury. It is thus hypothesized that blood flow of the PV‐BGM increases when superficial mucosal circulation is disturbed. The PV‐BGM can contribute to defensive mechanisms in the basal region of gastric mucosa. The abnormal healing process may disturb the defensive mechanism at the base of the gastric mucosa, thereby resulting in ulcer formation.

To examine the effect of vitamin C on blood flow in diabetic dental pulp, the animal model of streptozotocin (STZ)‐induced diabetic rats (i.v. injection of STZ 55 mg/kg BW) was used. Male Sprague‐Dawley rats weighing 200–250 g were divided into 3 groups: non‐diabetes (CON), diabetes (STZ), and diabetes supplemented by vitamin C (STZ+Vit C). Vitamin C was supplemented by drinking water (1 g/l). At 12 weeks (wks) and 24 wks after the STZ injection, the laser Doppler flow‐meter (Model ALF 21, USA) was used to measure pulpal blood flow (PBF) while the animals were anesthetized with sodium pentobarbital (50 mg/kg BW). The experimental results showed that at 12 and 24 wks after the STZ injection, hyperglycemia hypertension and loss of body weight were significantly developed. Simultaneously, decreased plasma vitamin C level was demonstrated significantly in STZ rats. The reduction of pulpal blood flow (PBF) in the lower incisors was observed in STZ rats at both monitored time points. Interestingly, the supplementation of vitamin C for 24 wks restored PBF. In conclusion, the present study demonstrated that long‐term supplementation of vitamin C, a natural antioxidant, could markedly prevent the diabetic‐induced reduction in PBF.

An imbalance between oxidative damage and antioxidative protection in association with the pathophysiology of atherosclerosis has been suggested. The aim of our study was to investigate the relationship between plasma lipids, the antioxidant system and oxidative damage in Thai patients with stable coronary artery disease (CAD). Sixty‐one patients (40 males, 21 females), who were angiographically defined as having CAD and were clinically stable, participated in this study. Thirty‐two healthy subjects (20 males, 12 females) served as normal controls. The investigation included the measurements of plasma lipid profiles and plasma total antioxidative status (TAS) such as plasma vitamin E erythrocyte glutathione (GSH) and glutathione peroxidase (GPx), as well as malondialdehyde (MDA) and total plasma total protein thiols (P‐SH). In patients with CAD, erythrocyte GSH and GPx were significantly lower than those found in controls. However plasma TAS and vitamin E were not significantly different between groups. Patients with CAD also had higher MDA and lower P‐SH levels than the controls, which represents the oxidative damage products of lipid and proteins. Multiple regression analysis revealed negative correlations between GSH and cholesterol, GSH and low density lipoprotein (LDL), vitamin E and MDA, as well as P‐SH and MDA. This study demonstrated the status of oxidative stress in patients with stable CAD. Since oxidative stress is the imbalance between the total oxidants and antioxidants in the body, any single oxidant/antioxidant parameter may not reflect the overall oxidative stress system. Thus, in patients with CAD, diets with various types of antioxidants may be more beneficial in increasing antioxidant activity than any particular antioxidant supplementation.

Endothelial function of cerebral microvessel in diabetes was evaluated using streptozotocin (STZ)‐induced diabetic rats (blood glucose of ≥300 mg/dl). At 36 weeks after STZ injection, the rats were anesthetized with sodium pentobarbital. The cerebral microcirculation in control and STZ groups was directly observed, using intravital fluorescence videomicroscopy. To evaluate the endothelial function in vivo, the number of leukocytes adhering to postcapillary venules were counted, and cerebral arteriolar responses to acetylcholine (ACh) and nitroglycerine (NTG) were examined. The results showed that the leukocyte adhesion to cerebral postcapillary venular endothelium increased significantly in STZ‐rats, compared with control rats. The vasodilatory responses of cerebral arterioles (20–30 μm) to ACh decreased significantly in STZ‐rats, compared with control rats (p<0.01), but the responses to NTG did not alter in diabetes. These results indicate that the impaired responses should occur on the endothelial cell. In conclusion, endothelial dysfunction induced in diabetes are characterized by impaired endothelium‐dependent vasodilation and increased leukocyte adhesion to endothelial cells.

Secondary cerebral ischemic injury is a major cause of mortality and disability from subarachnoid hemorrhage (SAH). In this study, the protective effects of nimodipine were investigated. Rat SAH models were divided into a sham‐operated group, a saline‐controlled, and a nimodipine‐treated group by an endovascular piercing method. Nimodipine, 100 μg/kg BW was injected intraperitoneally 30 minutes before operation and was repeated every 6 hours. Dynamic changes in cortical regional cerebral blood flow (rCBF) using a laser Doppler flow‐meter probe, and somatosensory evoked potentials (SEP) were estimated. Brain water content, sodium, potassium and calcium contents at different time points were determined. rCBF, latency of SEP, brain water and electrolyte contents did not statistically change in sham‐operated rats. In saline‐controlled rats, rCBF decreased immediately after SAH, and stabilized at low levels within 24 hours. The latency of SEP delayed gradually after SAH. Brain water and sodium increased, while potassium decreased at 6 hours and 24 hours. Brain calcium content increased significantly from 1 hour to 24 hours after induction of SAH. Extents of alterations of the above parameters caused by SAH in the nimodipine‐treated group were less than those in the saline‐controlled group, statistically. In conclusion, nimodipine partly prevents a decrease in cerebral blood supply and attenuates secondary cerebral ischemic injury after SAH.

Disputes on the significance of cerebral lymphatic drainage pathways under physiological and pathophysiological conditions still exist. The purpose of this experiment is to investigate the influence of cerebral lymphatic blockage on cerebral blood flow and cortex somatosensory evoked potential. Wistar rat cerebral lymphatic blockage models were established by removing cervical lymphatic nodes after obstructing their input and output tubes. Animals were divided randomly into a sham‐operated group and a cerebral lymphatic‐blockage group. Regional cerebral blood flow in different regions of the cortex were detected using a laser‐Doppler flowmeter probe, and cortex evoked potential was detected using an electromyogram and evoked potential instrument before the operation, then 1 day, 5 days and 7 days after the operation. Results showed that the sham operation had no obvious effect on regional cerebral blood flow and the latency of somatosensory evoked potential. From 1 day to 7 days after cerebral lymphatic obstruction, regional cerebral blood flow in different cortical regions decreased markedly (P<0.01). Latency of somatosensory evoked potential was significantly delayed on the 5th and 7th day after blockage of cerebral lymphatic drainage (P<0.01). We concluded that cerebral lymphatic drainage may play an important role in maintaining the equilibrium of the internal environment of the brain, and blockage of this pathway results in cerebral ischemia.

This study aimed to correlate plasmatic tissue‐type plasminogen activator (t‐PA) levels and activity with parameters of artery blood flow and vessel walls, nail fold microcirculation, hemorheology, serum glucose, and lipids. Thirty healthy volunteers (female/male 12/18) aged 40–60 (average 46) were included in the study. In citrate venous blood, the following parameters were determined: carotid mean velocity, carotid intimal‐medial‐thickness (IMT), capillary circulation parameters, hemorheology index, serum glucose, and lipids. Analysis of data showed that t‐PA concentration was positively and significantly correlated with total cholesterol, triglycerides, and serum glucose (P<0.05, P<0.05, and P<0.01), but t‐PA activity showed no correlation with them; among the hemorheology factors investigated, t‐PA concentration showed the strongest positive correlation with both whole blood viscosity and reduced blood viscosity at high and low shear rate separately (P<0.01), t‐PA activity showed no correlation with any hemorheology factors; t‐PA concentration showed no correlation with any investigated nail fold capillary parameters, whereas t‐PA activity was significantly and negatively associated with capillary loop number (P<0.05); t‐PA concentration and activity was not associated with values of carotid maximum intimal‐medial‐thickness (mIMT) and mean velocity or systolic, diastolic blood pressure (P>0.05). But subjects with mIMT 1.0 mm showed higher t‐PA levels compared with those with mIMT < 1.0 mm (P<0.05) and decreased carotid mean velocity (P<0.01). These findings suggest that multiple vascular disease risk factors would influence the t‐PA level; t‐PA concentration does not parallelize with t‐PA activity.

Diabetic maculopathy seen in the Philippines, specifically, the associated factors, the various lesions seen on fluorescein angiography, and the visual acuity associated with these lesions were characterized using 127 patients (254 eyes) with diabetic retinopathy based on the fluorescein angiography done at the Eye Referral Center in 1993. Results showed that 116 (91.34%) patients have maculopathy, the majority of which is bilateral (84.25%). Age (p=0.675), sex (p=0.357), hypertension (p=0.742), duration of diabetes (p=0.778) and myopia (p=0.742) were not significantly associated with maculopathy. However, severity of retinopathy (p=0.001) was significantly associated with it. Fluorescein angiographic findings are macular staining (83.86%), perifoveal capillary dropout or macular ischemia (10.76%), and preretinal traction and membrane (5.38%). Microaneurysm (72.65%) is the most common lesion associated with macular staining, followed by capillary leakage (4.04%), cystoid macular edema (3.59%), perifoveal capillary dropout with microaneurysm (2.24%), and capillary with microaneurysm leakage (1.34%). Exudates are associated with microaneurysm, perifoveal capillary dropout or a combination of the two. Vision was found to be marginally statistically different between the normal and maculopathy group (p=0.0505). The worst vision was seen in macular ischemia and preretinal traction and membrane, with mean visual acuity of 0.18 and 0.25, respectively. It is concluded that severity of retinopathy is the only variable significantly associated with maculopathy in this study. Good vision does not necessarily indicate a normal macula. Detailed examination and fluorescein angiography should be carried out, regardless of duration of diabetes.

This paper aimed at investigating the properties of cultured cardiomyocytes using microcirculatory and molecular technology to culture cardiomyocytes from different parts of the neonate Wistar rat's heart and record their spontaneous pulsation under time‐lapse video microscopy, then analyze their activity and inspect their survival rate and apoptotic rate under natural and nitric oxide conditions. The pulsation frequency in cardiomtocytes of different parts in heart are: 78.5±11.0 beats/min in the atrium, 88.4±6.3 beats/min in the left ventricle, 90.3±7.9 beats/min in the right ventricle and 115.3 ±11.4 beats/min in the cardiac apex, respectively, with an average frequency of 81.3 beats/min. Different concentrations of nitric oxide showed no effect on the frequency of cardiomyocyte pulsation. The survival rates of the above cardiomyocytes are 96.0%, 95.0%, 95.0%, and 95.3% respectively and 95.0% for the whole heart. The apoptotic rates are 1.3%, 1.1%, 4.8%, and 1.8% respectively and 5.1% for the whole heart. Different concentrations of nitric oxide had no effect on these results. Our study showed that cultured myocardial cells from different parts of the heart displayed various pulsation frequencies, and the frequency of the cardiac apex is the highest while the atrium is lowest. We also found that there is no statistically significant difference in the survival rates and apoptotic rates of different parts of the heart, and that nitric oxide has no effect on the beating frequency, survival rates or apoptotic rates of the cardiomyocytes in vitro.

This study was designed to observe the effect of tumor conditioned medium (TCM) on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to TCM from breast carcinoma cell line MDA‐MB‐231, then we measured their proliferation, apoptosis and cell cycle distribution by MTT and flow cytometery (FCM). Following the stimulation of TCM, HUVECs showed higher pro‐mitogenic and anti‐apoptotic ability than did the negative control group (ECGF‐free medium with 20% FBS), but a similar ability to the positive control group (medium with ECGF and 20% FBS). From these results, we can conclude that breast carcinoma cell line MDA‐MB‐231 could secret soluble pro‐angiogenic factors that induce HUVEC angiogenic switching, including cell cycle progression, proliferation and growth. The role and character of these factors remain to be further studied.

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA‐MB‐231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two‐dimensional polyacrylamide gel electrophoresis (2‐DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF‐free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 μg/ml ECGF and 20% FBS). Increased expression of cyclin D₁/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA‐MB‐231 may secrete soluble pro‐angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D₁/E increases and related proteins play an important role in this process.

The purpose of this study is to investigate the effect of L‐arginine (L‐Arg) on cerebral blood perfusion and vasomotion (perfusion motion) in microvessels following subarachnoid hemorrhage (SAH). Rat noncraniotomy SAH models were used and animals were divided into sham‐operated, saline‐treated, and L‐Arg‐treated groups. L‐Arg was injected intraperitoneally 30 minutes before the operation and repeated every 6 hours, with a single dose of 0.5 g/kg bw. Dynamic changes in regional cerebral blood flow (CBF) and vasomotion within 24 hours were measured using a laser Doppler flow‐meter probe. Serum nitric oxide (nitrite/nitrate) and plasma endothelin‐1 levels were also measured at different time points within 24 hours. Morphologic changes in neurons in the hippocampus CA1 region were examined. SAH gave rise to an immediate and persistent decrease in CBF in saline‐treated rats. Abnormal vasomotions with decreased frequency and amplitude were observed. Serum nitric oxide decreased, while plasma endothelin‐1 increased significantly. Neurons in the hippocampus CA1 region were severely damaged. The above pathological alterations in the L‐Arg‐treated group were alleviated. It was concluded that L‐Arg, which increases cerebral blood perfusion and improves vasomotions of microvessels by enhancing nitric oxide levels and decreasing endothelin‐1 levels in blood, exerts a protective effect on secondary cerebral ischemic injury following experimental SAH.

In the interest of creating readily transfusable blood, we have developed a method of covalently bonding methoxy polyethylene gylcol (mPEG) to the erythrocyte membrane to mask antigens which would otherwise cause immunorejection in unmatched blood transfusions. Previous studies have reported the ability of mPEG to mask erythrocyte antigens without significantly affecting viability, morphology, deformability, and other membrane functions. Our spectrophotometric analysis of modified erythrocytes has shown that as well as its other properties, mPEG‐coated red blood cells can still take up oxygen, while remaining antigenically silent.

Ghrelin, leptin and adiponectin are three hormones which are frequently associated with metabolism, obesity and appetite. Recently, it has been shown that they may possess other physiologic roles, specially in connection with the circulation. Ghrelin infusion increases forearm blood‐flow in a dose‐dependent manner. Leptin has been shown to be involved not only in thermogenesis but angiogenesis as well. Adiponectin, apart from its insulin‐sensitizing action, appears to modulate inflammation by inhibiting monocyte adhesion to endothelial cells. Six monkeys, which had been classified as being in the pre‐diabetic state, where administered a triglyceride lowering regimen. Microvascular function was assessed using a laser Doppler flow‐meter during a temperature provocation test. Percent change in flow from baseline following temperature elevation, as well as percent change in flow/degree rise in temperature were used to evaluate microvascular reserve and reactivity. Using univariate analysis, it appears that increased perfusion is significantly correlated with adiponectin, followed by leptin. Flow was also positively correlated with ghrelin, but the relationship did not attain significance. As expected, flow was also negatively and significantly correlated with fibrinogen. Trends show that flow was also negatively correlated to circulating triglyceride levels (p=0.08). The data indicate that the three hormones appear to possess microvascular actions that may impact on their other physiologic functions.

The maturity of pericytes in cerebral neocapillaries induced by two different growth factors: basic fibroblast growth factor (bFGF) and platelet‐derived growth factor (PDGF), was examined using an immunohistochemical staining technique. Cerebral angiogenesis was induced in mice by implanting a sandwich system of bFGF/PDGF gel and nylon‐mesh over the exposed cortex. On 28th day after incubation, a small volume of cerebral tissue with the nylon‐mesh was isolated and stained using tetramethyl rhodamine isothiocyanate (TRITC)‐labeled secondary antibody to the primary antibody against NG₂ proteoglycan and fluorescein isothiocyanate (FITC)‐labeled Griffonia simplicifolia (GS)‐lectin. Using a confocal laser microscopic system, we observed the cerebral neocapillaries on the upper surface of the nylon‐mesh and evaluated the maturity of pericytes stained with NG₂ based on the fluorescence immunohistological images. The pericyte appeared rich in neocapillaries induced by PDGF. It was suggested that pericytes might play a key role in the regulation of blood flow in neovessels.

To compare the level of endothelial nitric oxide synthase (eNOS) expression produced in heart and lung vascular tissue, the protein content was determined using Western blot analysis with the enhancement of image processing. Heart and lung extracts from 12 and 24 weeks from control (CON) and streptozotocin‐induced diabetic (DM) rats were collected for Western blot analysis. Using monoclonal antibody against rat eNOS protein (140 kDa), the eNOS‐protein bands were detected with enhanced chemiluminescence (ECL; Amersham) and exposured to film (Hyperfilm‐ECL; Amersham). Images of eNOS bands on each film were then scanned and saved to digital files. Using Global Lab Image software, the number of pixels in each digital file was counted and calibrated for eNOS‐protein content. For the CON and DM groups, the mean values of eNOS‐protein contents were calculated and expressed as a percentage of total protein content, 5 μg. It was found that the eNOS level in DM hearts was significantly decreased, as compared to age‐matched CON hearts. On the other hand, eNOS levels in DM lungs was increased, compared to CON lungs. Therefore, it may be concluded that high, not low, flow‐mediated eNOS expression is a good measure of hyperglycemic‐induced endothelial dysfunction.

The purpose of this study was to compare the short‐term effects of an intensive lifestyle modification (ILM) program on lipid peroxidation and antioxidant systems in patients with coronary artery disease (CAD). Twenty‐two patients in the control group continued to receive their conventional treatment with lipid‐lowering drugs, whereas 22 patients in the experimental group were assigned to intensive lifestyle modification (ILM) without taking any lipid‐lowering agent. The ILM program comprised dietary advice on low‐fat diets, high antioxidants and high fiber intakes, yoga exercise, stress management and smoking cessation. After 4 months of intervention, patients in the experimental group revealed a statistically significant increase in plasma total antioxidants, plasma vitamin E and erythrocyte glutathione (GSH) compared to patients in the control group. There was no significant change in plasma malondialdehyde (MDA), a circulating product of lipid peroxidation, in either group. We concluded that the ILM program increased circulating antioxidants and reduced oxidative stress in patients with CAD.

Decreased levels of nitric oxide play a role in the development of cerebral ischemia secondary to subarachnoid hemorrhage (SAH). The protective effect of L‐arginine on brain edema following SAH was investigated in this study. Rats were divided randomly into a sham‐operated, a SAH+saline group and a SAH+L‐arginine group. At different time points, brain water content was determined using the wet and dry weight compared method. Brain sodium content, potassium content and calcium content were detected using an atomic absorption spectral photometer. Somatosensory evoked potentials (SEP) were also detected. It was found that rat SAH models were successfully replicated. In the SAH+saline group, brain water and sodium content were significantly higher at 6 h and 24 h than those in the sham‐operated group, while brain potassium content was statistically lower than that in the sham‐operated group. Brain calcium content increased from 1 h to 24 h after induction of SAH. SEP latency progressively delayed. In the SAH+L‐arginine group, increases in brain water content, sodium content and calcium content, as well as decreases in brain potassium content, were not as obvious as in the SAH+saline group. L‐arginine partly prevented a delay in SEP latency. In conclusion, L‐arginine, a substrate of nitric oxide synthesis, may relieve brain edema in rats with experimental SAH.

A new technique using particle image velocimetry (PIV) has been developed to evaluate the detailed velocity profiles of red cells flowing in microvessels. The microcirculation in rat mesentery was directly observed using high‐speed videomicroscopy, and the images of red cells flowing in the mesenteric arterioles were recorded simultaneously with the arterial blood pressure. Based on the high‐speed videomicroscopic images obtained, velocity vectors in single or branched arterioles were evaluated to obtain velocity profiles across the cross‐section of arterioles. It was shown that in single and straight arterioles the velocity profile was blunt with a pit at the central region, and its pit was marked in bifurcation. The present technique enables us to analyze red cell velocity profiles up to 0.8 μm in the spatial resolution and 1 msec in the time interval.

The erythrocyte aggregation and deformability of blood samples obtained from normal subjects and malaria patients are determined by microscopic imaging and laser aggregometry techniques, and optical hemorheometer, respectively. By these techniques several parameters are determined but four parameters, aggregate sedimentation velocity (ASV), effective number of cells (ENC), process completion time (PCT) and mean filtration time (MFT), show significant variation in malaria patients compared to that of healthy subjects. For malaria severity analysis artificial neural network (ANN), based on feedforward‐error back‐propagation algorithm in a supervisory training mode is proposed. This network is first trained for different number of epochs ranging from 20 to 50 by set of patterns and at 30 epochs training session the minimum mean square error (MSE) between desired and actual output is obtained. By applying the same procedure the test patterns belonging to normal, non‐severe, severe, and highly severe malaria (NSM, SM and HSM) are identified. The results show that malaria with high severity is classified accurately (100%). The success of classification for non‐severe and mildly‐severe malaria ranges from 60% to 80%.

Glomerular endothelial cell dysfunction (GED) with defective release of vasodilator has been delineated in nephrosis (NS) in vivo and in vitro studies. In NS with focal segmental glomerulosclerosis (FSGS), an immunocirculatory balance may be impaired due to defective anti‐inflammatory cytokine. This study aimed at simultaneous determination of both proinflammatory cytokine (tumor necrosis factor alpha) and an anti‐inflammatory cytokine (interleukin‐10) in NS with FSGS. An endothelial cell cytotoxicity (ECC) was also examined using nephrotic serum. It was shown that (1) the initial endothelial cell cytotoxicity was significantly different from the control, (2) ratio between tumor necrosis alpha and interleukin‐10 was significantly elevated, and (3) intrarenal hemodynamics was changed significantly.