Perivascular Innervation: A Multiplicity of Roles in Vasomotor Control and Myoendothelial Signaling

The control of vascular resistance and tissue perfusion reflect coordinated changes in the diameter of feed arteries and the arteriolar networks they supply. Against a background of myogenic tone and metabolic demand, vasoactive signals originating from perivascular sympathetic and sensory nerves are integrated with endothelium‐derived signals to produce vasodilation or vasoconstriction. PVNs release adrenergic, cholinergic, peptidergic, purinergic, and nitrergic neurotransmitters that lead to SMC contraction or relaxation via their actions on SMCs, ECs, or other PVNs. ECs release autacoids that can have opposing actions on SMCs. Respective cell layers are connected directly to each other through GJs at discrete sites via MEJs projecting through holes in the IEL. Whereas studies of intercellular communication in the vascular wall have centered on endothelium‐derived signals that govern SMC relaxation, attention has increasingly focused on signaling from SMCs to ECs. Thus, via MEJs, neurotransmission from PVNs can evoke distinct responses from ECs subsequent to acting on SMCs. To integrate this emerging area of investigation in light of vasomotor control, the present review synthesizes current understanding of signaling events that originate within SMCs in response to perivascular neurotransmission in light of EC feedback. Although often ignored in studies of the resistance vasculature, PVNs are integral to blood flow control and can provide a physiological stimulus for myoendothelial communication. Greater understanding of these underlying signaling events and how they may be affected by aging and disease will provide new approaches for selective therapeutic interventions.     

Role of endothelin-1 induced by insulin in the regulation of vascular cell growth

Insulin is not only a growth factor for vascular cells, but also an inducer of other vasoactive substances such as endothelin-1 (ET-1) in vascular cells. The aim of the present study was to assess the role of endothelial cells (EC) in insulin mediated vascular smooth muscle cell (VSMC) proliferation. Cultured human aortic EC and VSMC were separately incubated. EC were stimulated with insulin (0 to 1000 microU/mL) for 24 h, in the presence or absence of anti-insulin-growth factor-1 (anti-IGF-1) receptor antibody (alphaIR(3)) or a nonselective ET-1 receptor antagonist (TAK044). Cell proliferation was measured by determining (3)H-thymidine uptake. Although 10 microU/mL insulin did not affect ET-1 production in the EC culture medium, a higher concentration of insulin stimulated it. Production of ET-1 in EC was activated by insulin via the IGF-1 receptor, inasmuch as alphaIR(3) blocked insulin mediated upregulation of ET-1. There was no significant difference in (3)H-thymidine incorporation in the presence of insulin (up to 1000 microU/mL) or TAK044. Culture medium from EC stimulated with insulin enhanced VSMC proliferation, which was almost totally suppressed by TAK044. Insulin induced VSMC growth dose dependently when VSMC were cultured alone. In contrast, insulin at concentrations of 100 microU/mL or lower failed to stimulate growth of co-cultured VSMC, but only at 330 microU/mL or higher concentrations stimulated VSMC growth in this system. Of interest, VSMC proliferation was greatest when L-NAME was added and co-cultured with EC. In summary, a severely hyperinsulinemic state may regulate VSMC and EC proliferation via activation of vasoactive substances such as ET-1 and nitric oxide induced by insulin.     

Oxygenating the microcirculation: the perspective from blood transfusion and blood storage

Tissue oxygen delivery depends on red blood cell (RBC) content and RBC flow regulation in the microcirculation. The important role of the RBC in tissue oxygenation is clear from anaemia and the use of RBC transfusion which has saved many lives. Whether RBC transfusion actually restores tissue oxygenation is difficult to determine due to the lack of appropriate clinical monitoring techniques. Some patients with restored haemoglobin levels and stable haemodynamics still develop tissue hypoxia, emphasizing that, in addition to global parameters, local microcirculatory control mechanisms are also important in the restoration of tissue oxygenation. Both clinical and animal experimental studies have indicated that storage of RBC diminishes their ability to oxygenate the tissue. Several intrinsic RBC parameters that change during storage and might influence tissue oxygenation will be mentioned. The release of vasodilators from RBC that will alter blood flow during hypoxia, mediated by haemoglobin in the RBC that functions as an oxygen sensor, could be impaired during storage. A better understanding of hypoxia-induced vasodilator release from RBC might become a potential target for drug development and improve tissue oxygenation after transfusion.     

Automated method for tracking individual red blood cells within capillaries to compute velocity and oxygen saturation

OBJECTIVE: The authors present a new method to track individual red blood cells (RBCs) as they move through capillaries. This method uses a recently developed Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) and the concept of space-time images to track RBCs between consecutive frames of video recordings of the microcirculation. METHODS: A space-time image displays in a single static image for a single capillary the location of all RBCs as a function of time. Analysis is performed on video tapes of RBC flow through capillaries to obtain velocity of individual cells as they traverse the capillary of interest. A space-time image is generated to track RBCs from one frame to the next and their velocities are computed. Based on the optical density values of each cell obtained from synchronized videotapes at two wavelengths, the oxygen saturation of a cell can be determined. In this manner, oxygen saturation can be tracked for the same cells as they move through the capillary. RESULTS AND CONCLUSIONS: These measurements, taken together, allow one to determine how much and how fast oxygen is being delivered to the surrounding tissue. This method provides, for the first time, a way to track individual RBCs flowing through capillary networks and study their RBC dynamics and oxygenation.     

Acute endothelial cell contraction in vitro: a comparison with vascular smooth muscle cells and fibroblasts.

The contractile responses of cultured rat and calf endothelial cells (EC), vascular smooth muscle cells (VSMC), and fibroblasts (FB) to vasoactive mediators (thrombin, serotonin, bradykinin, and histamine), forskolin, and cytochalasin B were compared. Cells were grown on a pliable silicone membrane, and contraction was assessed, using time-lapse video microscopy, by recording changes in the wrinkling of the silicone as the cells exerted tension on the surface. We found that all cells contracted in the presence of serum or thrombin and that VSMC and FB also contracted with serotonin stimulation. Bradykinin and histamine were not contractants in this system. Discrepancies between these results and reports of changes in permeability of endothelial layers in vitro and in vivo may be due to (1) the vascular segment from which EC were studied or (2) the possibility that certain mediators may provoke a noncontractile response that results in gap formation. Thus changes in vascular permeability, which occur during inflammation, may have both contractile and noncontractile components. Forskolin, known to indirectly inhibit myosin light-chain kinase activity, and cytochalasin B were potent relaxants, suggesting a similar smooth muscle-like contractile mechanism for all three cell types.     

Endothelial cell interactions with sickle cell, sickle trait, mechanically injured, and normal erythrocytes under controlled flow

Increased adhesive forces between sickle erythrocytes and endothelial cells (EC) have been hypothesized to play a role in the initiation of vasoocclusion in sickle cell anemia. Erythrocyte/human umbilical vein EC interactions were studied under controlled flow conditions for normal (AA), homozygous sickle cell (SS), sickle cell trait (AS), mechanically injured normal, and "high-reticulocyte control" RBC by using video microscopy and digital image processing. The number of adherent RBC was determined at ten-minute intervals during a washout period. Results indicate that SS RBC were more adherent than AA RBC. Mechanically injured (sheared) AA RBC were also more adherent than control normal cells but less adherent than SS RBC. AS RBC did not differ significantly in their adhesive properties from normal RBC. Less- dense RBC were more adherent to EC than dense cells for normal, SS, and high-reticulocyte control RBC. The number of cells adherent at a given time during washout was a very strong function of wall shear rate. In addition, at all shear rates studied, the average velocity of individual SS RBC in the region near the EC surface was approximately half that of AA RBC at the same bulk volumetric flow rate through the flow chamber. These findings suggest that the increased adhesion of sickle RBC is at least partially related to the increased numbers of less-dense RBC present. Increased adherence of the less-dense cells to the EC lining vessel walls could contribute to microvascular occlusion by lengthening vascular transit times of other sickle cells.     

Heterocellular contact at the myoendothelial junction influences gap junction organization

Heterocellular communication between vascular smooth muscle cells (VSMC) and endothelial cells (EC) at the myoendothelial junction (MEJ) is a critical part of control of the arteriolar wall. We have developed an in vitro model of the MEJ composed of primary cultures of murine EC and VSMC. Immunoctytochemistry and immunoblots demonstrated Cx37 and Cx43 in both cell types, whereas Cx40 was found only in EC. Cx37 was excluded from the MEJ in both EC and VSMC. Connexin composition as well as functionality of the gap junctions at the MEJ was assessed by measuring diffusional transfer of biocytin and Cy3. Using connexin-specific blockers and manipulations of expression of individual connexin proteins, we confirmed that Cx37 is not a part of EC-VSMC coupling, and we demonstrated that heterotypic gap junctions are functional at the MEJ. We speculate that specific gap junction organization may be a vital component of EC-VSMC contact at the MEJ.     

Ex-vivo red blood cells generation: a step ahead in transfusion medicine?

Blood transfusion and especially red blood cell (RBC) transfusion is an essential therapeutic act because it might be life-saving in several situations such as massive bleeding or as prolonged quality-of-life therapies in patients with chronic anemic disorders. Although the need for blood is thereby widespread, there is however a major imbalance between demand and supply of donors, so that there is a mounting research to develop suitable surrogates for human donated blood. Functional RBCs have already been generated from a variety of cellular progenitors (i.e., somatic stem cells, human embryonic stem cells and induced pluripotent stem cells) and synthetic biomaterials. Although these types of “artificial blood” carry several advantages over donated blood, including larger supply, lower risk of blood-borne pathogens transmission, no risk of immune incompatibility using group O, RhD-negative RBCs, and extended survival of stored RBCs, their efficacy has not been extensively tested thus far in clinical trials. Therefore, while it seems premature at this point in time to conclude that ex-vivo manufacturing of blood might be the definitive solution to the current shortage of blood supply, it represents however a valuable starting point for translational research in transfusion medicine.     

The Role of Red Blood Cell Exchange Transfusion in the Treatment and Prevention of Complications of Sickle Cell Disease

Abstract: Patients with sickle cell disease have abnormal red blood cells (RBCs). This can cause chronic hemolytic anemia and vaso‐occlusion leading to tissue hypoxemia and organ dysfunction. RBC exchange transfusion can, without increasing the whole‐blood viscosity, quickly replace abnormal erythrocytes with normal and raise the hematocrit resulting in improved delivery of oxygen to hypoxic tissues. Unfortunately, transfusion can also be associated with complications. This paper reviews the role of transfusion, both simple and exchange, in the treatment and prevention of sickle‐related complications. The benefits of exchange versus simple transfusion and transfusion versus alternative therapies are discussed.     

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34⁺ cells using a four-phase culture system. We first cultured CB CD34⁺ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34⁺ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34⁺ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 10¹² RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 10¹³ RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.     

Some characteristics of human red blood cells separated according to their size: a comparison with density-fractionated red blood cells

During their in vivo ageing, red blood cells (RBC) increase in density and become smaller. Age-defined RBC subpopulations are usually collected by centrifugation. A fractionation according to RBC volume has been proposed as an improved alternative to such age separation. Because a few data reported in the literature indicate some discrepancies between the two methods, blood samples were separated either by centrifugation or by counterflow centrifugation, and some characteristics of the RBC thus fractionated were studied. The enzyme activities decrease either when the density rises or when the volume (MCV) decreases. However, the comparison of other RBC characteristics strongly suggests that these two procedures do not lead to the collection of the same RBC subpopulations: for instance, the hemoglobin content increases when the MCV rises, whereas it remains constant whatever the RBC density is. With radiolabelled cells, it is shown 1) that the most dense RBC are recovered in all the size-separated RBC subpopulations, even though they tend to concentrate in the fractions with the largest MCV, and 2) that the smallest RBC are almost fairly distributed in all the RBC subpopulations, whatever their density, whereas the largest RBC are mainly, but not exclusively, present in the high-density fractions. Thus, fractionation according to size does not match separation according to density. Taken together with results from in vivo experiments carried out in mice and with the fact that reticulocytes are present in all the size-separated fractions, these data suggest that counterflow centrifugation may be a very questionable procedure to achieve a RBC fractionation according to age and therefore that RBC volume might not be a reliable criterion of RBC age.     

The role of the kidney in blood volume regulation: the kidney as a regulator of the hematocrit

The kidney plays a pivotal role in the regulation of blood volume by controlling the plasma volume and red blood cell (RBC) mass. Further, it is proposed that the kidney coordinates the relative volumes of these 2 blood components and in so doing regulates the hematocrit. This novel function as proposed is a functional concept whereby the kidney does not simply produce erythropoietin, but that the kidney regulates the hematocrit is termed the critmeter function. The kidney is unique in that it can indirectly report on blood volume as a tissue oxygen signal. It is proposed that the kidneys detect small changes in tissue oxygen tension for erythropoietin production at the critmeter, a functional unit of marginal oxygen tension within the kidneys. As the production of erythropoietin is modulated by angiotensin II, the renin-angiotensin system entrains the production of erythropoietin as part of the effector signals of the feedback loop of blood volume regulation. Collectively, the consideration of these points generates a paradigm shift in our understanding of blood volume regulation in that the role of the kidney may be expanded from simply "producing" erythropoietin to regulating the hematocrit. Further, this concept broadens the scope of the traditionally identified effector mechanisms of plasma volume regulation to include the modulation of erythropoietin production and hence RBC mass. The inclusion of both plasma volume and RBC mass as factors targeted by the effector signals recapitulates that whole blood volume is sensed and reported in the afferent signals. In summary, distinct sensing and effector mechanisms for regulating the volume of the two components of whole blood (plasma and red cell mass) are recognized. The coupling of the regulation of these 2 components of blood volume is highlighted.     

Human urotensin-II potentiates the mitogenic effect of mildly oxidized low-density lipoprotein on vascular smooth muscle cells: comparison with other vasoactive agents and hydrogen peroxide.

Human urotensin-II (U-II) is the most potent vasoactive peptide identified to date, and may be involved in hypertension and atherosclerosis. We investigated the effects of the interactions between U-II or other vasoactive agents and mildly oxidized low-density lipoprotein (mox-LDL) or hydrogen peroxide (H2O2) on the induction of vascular smooth muscle cell (VSMC) proliferation. Growth-arrested rabbit VSMCs were incubated with vasoactive agents (U-II, endothelin-1, angiotensin-II, serotonin, or thromboxane-A2) in the presence or absence of mox-LDL or H2O2. [3H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. On interaction with mox-LDL or H2O2, U-II induced the greatest increase in [3H]thymidine incorporation among these vasoactive agents. A low concentration of U-II (10 nmol/l) enhanced the potential mitogenic effect of low concentrations of mox-LDL (120 to 337%) and H2O2 (177 to 226%). U-II at 50 nmol/l showed the maximal mitogenic effect (161%), which was abolished by G protein inactivator (GDP-beta-S), c-Src tyrosine kinase inhibitor (radicicol), protein kinase C (PKC) inhibitor (Ro31-8220), extracellular signal-regulated kinase (ERK) kinase inhibitor (PD98059), or Rho kinase inhibitor (Y27632). Mox-LDL at 5 microg/ml showed the maximal mitogenic effect (211%), which was inhibited by free radical scavenger (catalase), intracellular and extracellular antioxidants (N-acetylcysteine and probucol), nicotinamide adenine dinucleotide phosphate oxidase inhibitor (diphenylene iodonium), or c-Jun N-terminal kinase (JNK) inhibitor (SP600125). These results suggested that U-II acts in synergy with mox-LDL in inducing VSMC DNA synthesis at the highest rate among these vasoactive agents. Activation of the G protein/c-Src/PKC/ERK and Rho kinase pathways by U-II together with the redox-sensitive JNK pathway by mox-LDL may explain the synergistic interaction between these agents.     

A new video image analysis system to study red blood cell dynamics and oxygenation in capillary networks

OBJECTIVE: The authors present a Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) to study red blood cell (RBC) dynamics and oxygenation in capillary networks. The system enables analysis of capillaries to study geometry and morphology and provides values for capillary parameters such as diameter and segment length. It also serves as an analysis tool for capillary RBC flow characteristics, including RBC velocity, lineal density, and supply rate. Furthermore, the system provides a means of determining the oxygen saturation of hemoglobin contained within RBCs, by analysis of synchronized videotapes containing images at two wavelengths, enabling the quantification of the oxygen content of individual RBCs. METHODS: Video recordings of RBC flow at two wavelengths, 420 nm (isosbestic) and 436 nm (oxygen sensitive), are made using a dual camera video microscopy system. The 420-nm recording is used to generate images based on the variance of light intensity fluctuations that help to identify capillaries in a given field of view that are in sharp focus and exhibit flow of individual RBCs separated by plasma gaps. A region of interest enclosing the desired capillary is defined and a fixed number of successive video frames at the two wavelengths are captured. Next a difference image is created, which delineates the RBC column, whose width is used to estimate the internal diameter of the capillary. The 420-nm images are also used to identify the location and centroid of each RBC within the capillary. A space-time image is generated to compute the average RBC velocity. Lineal density is calculated as the number of RBCs per unit length of a capillary segment. The mean optical density (OD) of each RBC is calculated at both wavelengths, and the average SO(2) for each cell is determined from OD(436)/OD(420). RESULTS AND CONCLUSIONS: MASCOT is a robust and flexible system that requires simple hardware, including a SGI workstation fitted with an audio-visual module, a VCR, and an oscilloscope. Since the new system provides information on an individual cell basis from entire capillary segments, the authors believe that results obtained using MASCOT will be more accurate than those obtained from previous systems. Due to its flexibility and ease of extension to other applications, MASCOT has the potential to be applied widely as an analysis tool for capillary oxygen transport measurements.     

Red blood cell intercellular interactions in oxidative stress states

Red blood cell (RBC) intercellular interactions, i.e., self-aggregation and adherence to endothelial cells (EC), play important roles in microcirculation. These RBC flow properties are determined by cell membrane components, which are prone to damaging reactive oxygen species (ROS) produced in oxidative stress (OS) states. Alterations in RBC aggregability and adherence have been linked to the pathophysiology in numerous diseases associated with OS. We investigated RBC intercellular interactions in four OS states--thalassemia, treatment of RBC with phenyl-hydrazine or H2O2, and photodynamic virus inactivation of blood units. All these OS states increased RBC adherence to EC, but only part of them elevated their aggregability, while others abolished it. It is proposed that (1) different OS states might induce disparate effects on RBC intercellular interactions; (2) RBC aggregability and adherence to EC, although both intercellular interactions, are controlled by different cell surface factors.     

Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease

The deformability of erythrocytes is a critical determinant of blood flow in microcirculation. By capturing red blood cells (RBC) with optical tweezers and dragging them through a viscous fluid we were able to measure their overall elasticity. We measured, and compared, the RBC deformability of 15 homozygous patients (HbSS) including five patients taking hydroxyurea (HU) for at least 6 months (HbSS/HU), 10 subjects with sickle cell trait (HbAS) and 35 normal controls. Our results showed that the RBC deformability was significantly lower in haemoglobin S (HbS) subjects (HbSS and HbAS), except for HbSS/HU cells, whose deformability was similar to the normal controls. Our data showed that the laser optical tweezers technique is able to detect differences in HbS RBC from subjects taking HU, and to differentiate RBC from normal controls and HbAS, indicating that this is a very sensitive method and can be applied for detection of drug-response in sickle cell disease.     

Red blood cell transfusion in clinical practice

Every year, about 75 million units of blood are collected worldwide. Red blood cell (RBC) transfusion is one of the few treatments that adequately restore tissue oxygenation when oxygen demand exceeds supply. Although the respiratory function of blood has been studied intensively, the trigger for RBC transfusion remains controversial, and doctors rely primarily on clinical experience. Laboratory assays that indicate failing tissue oxygenation would be ideal to guide the need for transfusion, but none has proved easy, reproducible, and sensitive to regional tissue hypoxia. The clinical importance of the RBCs storage lesion (ie, the time-dependent metabolic, biochemical, and molecular changes that stored blood cells undergo) is poorly understood. RBCs can be filtered, washed, frozen, or irradiated for specific indications. Donor screening and testing have dramatically reduced infectious risks in the developed world, but infection remains a major hazard in developing countries, where 13 million units of blood are not tested for HIV or hepatitis viruses. Pathogen inactivation techniques are in clinical trials for RBCs, but none is available for use. Despite serious immunological and non-immunological complications, RBC transfusion holds a therapeutic index that exceeds that of many common medications.     

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro

Background

The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes.

Design and Methods

To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro.

Results

The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes.

Conclusions

The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.     

Red blood cell alloimmunization is influenced by recipient inflammatory state at time of transfusion in patients with sickle cell disease

Sickle cell disease (SCD) patients are at increased risk of red blood cell (RBC) alloimmunization. Recipient inflammatory state at time of transfusion has been shown to regulate alloimmunization in murine models, but evidence is lacking in SCD patients. We retrospectively studied a cohort of alloimmunized SCD patients to determine the influence of pro‐inflammatory SCD‐related complications at time of transfusion on alloimmunization. For each transfusion, the presence of pro‐inflammatory state, degree of RBC antigen matching, unit age, storage solution and alloantibody detection date were ascertained. Transfusion‐associated pro‐inflammatory events were compared between transfusions resulting and not resulting in new alloantibodies. Univariate analysis and multivariate logistic regression were performed. Fifty‐two patients received 3166 pre‐storage leuco‐reduced transfusions of which 128 resulted in alloantibodies. Transfusions during inflammatory events were associated with increased alloantibody risk on univariate and multivariate analysis; acute chest syndrome and vaso‐occlusive crisis showed strongest associations with alloimmunization. Increased antigen matching demonstrated a protective effect on alloimmunization (univariate and multivariate analysis). Although an association was seen between citrate‐phosphate‐dextrose (adenine) stored units and alloimmunization on univariate analysis, no effect was found on multivariate analysis. Identifying recipient pro‐inflammatory states at time of transfusion that promote alloimmunization can impact RBC unit selection decisions for SCD patients at risk for alloimmunization.     

Insulin and the vasculature

Under physiologic conditions, insulin plays a vasculoprotective role by acting as a vasodilator through the stimulation of nitric oxide synthesis and release from the endothelium. In addition, insulin may decrease the contractile response of vascular smooth muscle cells (VSMC) to vasoactive agents by decreasing the intracellular calcium concentration [Ca²⁺]i. However, in the insulin resistance syndrome, the vasodilator effect of insulin may be blunted, an abnormality that may be related to endothelial dysfunction. Insulin resistance correlates with carotid wall thickness and stiffness, but the relationship is modified by sex and ethnic factors. Insulin can act as growth factor stimulating VSMC growth in culture. Insulin-sensitizing agents are efficacious in improving the vascular pathology associated with insulin resistance.