Rp-cAMPS has ho effect on adenosine A1 receptors in guinea-pig cardiomyocytes

Rp-cAMPS, a protein kinase A inhibitor, is used in the investigation of the cAMP-dependent systems. A report by Musgrave et al. (11) has suggested that Rp-cAMPS may also act on adenosine receptors. To determine whether this occurs in guinea-pig ventricular myocytes, Rp-cAMPS was applied in the presence and absence of DCPCX, an adenosine A₁ receptor antagonist. The isoprenaline-induced response was significantly decreased by Rp-cAMPS and the effect was not altered by the presence of DCPCX. Therefore Rp-cAMPS has no effect on cell contraction via adenosine A₁ receptors and can reliably be used to investigate cyclic AMP-dependent systems in isolated cardiac myocytes. Received: 23 April 1999, Returned for 1. revision: 26 May 1999, 1. Revision received: 23 August 1999, Returned for 2. revision: 28 September 1999, 2. Revision received: 15 November 1999, Accepted: 17 November 1999     

Transient regulation of transport by pericytes in venular microvessels via trapped microdomains

A phenomenon that has defied explanation for two decades is the time scale for transient reabsorption in the classic experiments of Michel and Phillips on individually perfused frog mesentery microvessels. One finds that transient reabsorption lasts <2 min before a new steady state of low filtration is established when the lumen pressure is abruptly dropped from a high to a low value. Our experiments in frog and rat venular microvessels under a variety of conditions revealed the same time trend for new steady states to be established as in Michel and Phillips' experiments. In contrast, one theoretically predicts herein that the time required for the tissue albumin concentration to increase to values for a new steady state to be achieved through reabsorption is in the order of several hours. In this paper we propose a new hypothesis and theoretical model for this rapid regulation, namely that pericytes covering the interendothelial cleft exits create small trapped microdomains outside the cleft exits which regulate this transient behavior. Our electron microscopy studies on rat mesenteric venular microvessels reveal an average pericyte coverage of approximately 85%. The theoretical model based on this ultrastructural study predicts an equilibration time on the order of 1 min when the lumen pressure is abruptly lowered. The basic concept of a trapped microdomain can also be extended to microdomains in the interstitial space surrounding skeletal muscle capillaries.     

Platelet factor-4 excretion in myeloproliferative disease: implications for the aetiology of myelofibrosis

S ummary. Experimental evidence suggests that the fibroblastic proliferation often associated with the myeloproliferative disorders is not part of the neoplastic process, but is secondary to an unknown stimulus. This stimulus may be a factor derived from platelets which promotes the proliferation of fibroblasts in vitro (PDGF). Plateletderived growth factor is localized to platelet α-granules together with PF4 and β-TG. As an indicator of α-granule release, we have measured PF4 levels in plasma. platelets and urine in 46 normal subjects and 49 patients with myeloproliferative disorders, secondary thrombocytosis and miscellaneous malignancies. All 11 patients with elevated urinary PF4 excretion exhibited myelofibrosis, whereas 11 of 22 patients with documented myelofibrosis had urinary PF4 excretion in the normal range. No correlation was seen between marrow fibrosis and plasma levels or the platelet content of PF4. The data are consistent with the possibility that release of mitogen(s) from platelet or megakaryocyte α-granules in some patients with myeloproliferative disorders is pathogenetically related to the development of marrow fibrosis.     

Trophoblast functions, angiogenesis and remodeling of the maternal vasculature in the placenta

One of the most important local adaptations to pregnancy is the change in maternal blood flow to the implantation site. In rodents and primates, new blood vessels form through angiogenesis, dilate and then become modified such that the blood enters into trophoblast cell-lined sinuses (hemochorial). Evidence from gene knockout mice suggests that factors from the placenta regulate the uterine vasculature. Consistent with this, trophoblast giant cells produce a number of angiogenic and vasoactive substances that may mediate these effects. Teratocarcinomas containing large numbers of trophoblast giant cells (derived from Parp1 gene-deficient ES cells) show similar 'hemochorial' host blood flow, implying that the effects are not specific to the uterine vascular bed. As in primates, murine trophoblast cells also invade into the uterine arteries of the mother. However, in normal pregnancy, dilation of the uterine arteries may be largely mediated by the effect of uterine natural killer cells.     

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA₂, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA₂ and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood–retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA₂ inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA₂ inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA₂ may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.Breakdown of the blood–retinal barrier (BRB) is a significant pathophysiological event during diabetic retinopathy (DR). Leakage of plasma proteins and lipids into the neuropile form exudates, whereas excessive vasopermeability can lead to diabetic macular edema (DME), which is a major cause of vision loss (1). Both DME in patients and BRB dysfunction in animal models have been linked to inflammatory processes, endothelial dysfunction, and compromise of normal neuroglial–vascular interactions (2). A number of therapeutic options are available to treat DME. Laser photocoagulation is effective in slowing the progression of DME; however, it carries risk of serious adverse side effects including visual field defects, retinal scarring, and foveal burns. Intravitreal injection of antiinflammatory corticosteroids has proven value, but this intervention also carries significant side effects including cataract formation and elevated intraocular pressure. More recently, neutralization of vascular endothelial growth factor (VEGF) in the retina has become a mainstream treatment of DME (3) although this is not effective for all DME patients (4). Moreover, repeated intravitreal injections can result in some patients becoming refractory to VEGF blockade (5), which suggests that other mechanisms also drive vascular permeability during DME. Concerns have also been raised that targeting the VEGF pathway could inadvertently compromise retinal neuroglial and microvascular survival (6) although, thus far, no evidence of these outcomes have been observed in clinical trials of anti-VEGF for DME. Nevertheless there is a need for additional therapeutic approaches to treat DME, either alone or adjunctively with currently available therapeutics.An alternative permeability-inducing agent in diabetic retina could be lysophosphatidylcholine (LPC), which is elevated in plasma of diabetic patients (7) and has demonstrated permeability-enhancing activity in cultured nonneural endothelial cells (ECs) (8). The principal enzyme responsible for the production of LPC is a calcium-independent phospholipase A₂ called lipoprotein-associated phospholipase A₂ (Lp-PLA₂) (also known as platelet-activating factor acetylhydrolase or type VIIA PLA₂). Lp-PLA₂ can elicit a broad range of proinflammatory and proapoptotic effects in blood vessels related to its hydrolysis of modified polyunsaturated fatty acids within oxidized low-density lipoprotein (oxLDL) into LPC and oxidized nonesterified fatty acids (OxNEFA) (9). Elevated Lp-PLA₂ has been proposed as a predictive biomarker in stroke (10), atherosclerosis (11), and coronary heart disease (12). Macrophages and other proinflammatory cells are a primary source of Lp-PLA₂ in the systemic circulation (13), although endothelial cells also express this enzyme (14).Darapladib, a specific inhibitor of Lp-PLA₂, has been shown to reduce atherosclerosis in both diabetic/hypercholesterolemic pigs (15) and ApoE-deficient mice (16). In diabetic/hypercholesterolemic pigs, darapladib protected against blood–brain barrier (BBB) dysfunction and vascular permeability (17). Darapladib has been studied in nearly 16,000 patients with coronary heart disease, and approximately one-third of this study population had diabetes mellitus (18). In a further study, a 3-month daily treatment with 160 mg of darapladib orally showed reduction of DME and an improvement in visual acuity in patients (19). These observations suggest that Lp-PLA₂ and its mechanism of action warrant further investigation in the context of DR. In this study, we have studied the inhibition of Lp-PLA₂ in diabetic rats with regards to BRB function and assessed the effect of LPC on neural microvascular endothelial cells. We show inhibition of Lp-PLA₂ was effective in reducing diabetes-induced retinal vasopermeability and that the effect of this inhibition was additive with VEGF neutralization, suggesting this enzyme could be an efficacious therapeutic target for DME, either alone or in combination with anti-VEGF therapeutics.     

Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion.

Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null cells were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.     

Somatostatin reduces posthypoglycemic insulin resistance in insulin-dependent diabetes mellitus

In order to study whether somatostatin reduces posthypoglycemic insulin resistance, hypoglycemia was induced between 7.00 and 8.00 h by an iv infusion of insulin with and without somatostatin (250 micrograms/h) in 6 male patients with insulin-dependent diabetes mellitus (IDDM). Exogenous glucagon was infused to substitute for the suppression of its endogenous release (1.0-1.5 ng.kg-1.min-1). Insulin resistance was assessed by a somatostatin-insulin-infusion test (SIGIT) between 11.00 and 15.00 h. In the study without hypoglycemia, blood glucose was kept close to 6 mmol/l from 8.00 h until start of the SIGIT. In both hypoglycemic studies similar nadir blood glucose levels were achieved and hypoglycemia evoked the same increase of plasma epinephrine and cortisol, whereas plasma glucagon remained at its basal level. The growth hormone response to hypoglycemia was suppressed by somatostatin. At the onset of the SIGIT, the plasma levels of the counterregulatory hormones had returned to basal, and blood glucose and plasma free insulin concentrations were almost identical. During the SIGIT there were no differences in plasma free insulin or counterregulatory hormone levels. Insulin resistance, as seen following hypoglycemia, was not demonstrable in the study with somatostatin. It is concluded that somatostatin reduces insulin resistance following hypoglycemia in patients with IDDM. It is therefore suggested that an analogue with a specific GH release inhibiting property may be useful in reducing glycemic instability when given as adjunct therapy to insulin in patients with labile glycemic control.     

Did maternal mortality ratio increase in Malawi between 1992-1998? Review of Malawi Demographic and Health Surveys and other data sources

Despite various programmes aimed at reducing the maternal mortality ratio (MMR) and improving reproductive health globally, and in Malawi especially, the 2000 Malawi Demographic and Health Survey (DHS) reported an MMR for Malawi as 1221 deaths per 100 000 live births. This represented an almost 80% rise from the 620 maternal deaths/100 000 live births estimated in the 1992 DHS. The possible reasons behind the rise in the MMR include: the growing HIV/AIDS pandemic in Malawi with an estimated infection rate of 14%; and the deteriorating healthcare situation and inherent inaccuracies in the estimation of maternal mortality. Continued surveillance and identification of factors responsible for the deterioration of Malawi's MMR are suggested. It is necessary to design, implement and evaluate corrective measures in order to improve the situation.     

Right and left ventricular function after cardiac transplantation. Changes during and after rejection

BACKGROUND. Attempts to identify noninvasive markers of ventricular dysfunction accompanying acute rejection have been hampered by a lack of detailed simultaneous hemodynamic data. Therefore, we prospectively performed serial monitoring of detailed left and right heart hemodynamic parameters in cardiac transplant recipients at the time of routine endomyocardial biopsy to better define the physiology of the allograft heart during and after acute rejection. METHODS AND RESULTS. To better assess the pathophysiology of the rejection process, 18 cardiac transplant patients were prospectively studied by serial right heart micromanometer catheterization and digital image processing at the time of routine endomyocardial biopsy. Eleven patients had 18 episodes of rejection. Studies of baseline (negative biopsy preceding rejection), rejection (acute moderate rejection), and resolved (first negative biopsy after rejection) states were compared. Seven patients who did not experience an episode of rejection served as the control group. Right ventricular minimum and end-diastolic pressures increased from baseline values of 0.9 +/- 3.2 and 6.9 +/- 3.7 mm Hg, respectively, to 3.2 +/- 5.5 and 9.9 +/- 6.6 mm Hg, respectively, with rejection (both variables, p less than 0.05) and remained elevated despite histological resolution of rejection (4.3 +/- 5.5 and 10.0 +/- 7.1 mm Hg, respectively; p less than 0.05 for both variables compared with baseline values). Concurrently, right ventricular end-diastolic volumes (133 +/- 29, 119 +/- 27, and 114 +/- 30 ml; baseline, rejection, and resolved, respectively) and left ventricular end-diastolic volumes (133 +/- 24, 117 +/- 20, and 113 +/- 30 ml; baseline, rejection, and resolved, respectively) significantly decreased during rejection and remained decreased after resolution of rejection (rejection and resolved compared with baseline values, p less than 0.05). Right ventricular chamber stiffness (0.055 +/- 0.035, 0.085 +/- 0.057, and 0.092 +/- 0.076 mm Hg/ml; baseline, rejection, and resolution, respectively; rejection and resolved compared with baseline values, p less than 0.05) increased with rejection and remained elevated after resolution of rejection. Right ventricular peak filling rate also increased from a baseline value of 2.48 +/- 0.45 to 2.76 +/- 0.63 ml end-diastolic volumes per second with rejection (p less than 0.05). Elevation of right ventricular filling pressures, peak filling rate, and chamber stiffness with a concomitant decrease in end-diastolic volume is consistent with a restrictive/constrictive physiology. Mean arterial blood pressure and systemic vascular resistance were elevated after the resolution of rejection (compared with either rejection or baseline values, p less than 0.05) associated with a higher mean daily dose of prednisone (resolved compared with either baseline or rejection values, p less than 0.05). The control group experienced a time-dependent increase in mean and diastolic systemic arterial pressures (both comparisons, p less than 0.05) without detectable diastolic dysfunction. CONCLUSIONS. Persistence of biventricular diastolic dysfunction may be due to an irreversible effect of rejection, although multifactorial changes in left ventricular afterload occur that may complicate serial assessment of ventricular function.