Jennell White

Macrophages induce the adhesion phenotype in normal peritoneal fibroblasts

OBJECTIVE To determine whether macrophages, exposed to hypoxia, stimulate primary cultures of fibroblasts to acquire the adhesion phenotype. The adhesion phenotype has been previously characterized, in part, by increased fibroblast expression of transforming growth factor (TGF) β1, vascular endothelial growth factor (VEGF), and type I collagen. DESIGN Media collected from human macrophages cultured under hypoxic conditions (2% O(2)) were used to treat human peritoneal fibroblasts. Additionally, human peritoneal fibroblasts were treated with varying concentrations of TGF-β1. Real-time reverse-transcription polymerase chain reaction and Western blot analysis were used to measure mRNA and protein levels, respectively, for select adhesion markers: TGF-β1, VEGF, and, type I collagen. We hypothesized that macrophage secretion, under hypoxic conditions, is responsible for inducing the adhesion phenotype in human peritoneal fibroblasts. SETTING University research laboratory. PATIENT(S) Human macrophages and peritoneal fibroblasts. INTERVENTION(S) Macrophage-fibroblast interaction. MAIN OUTCOME MEASURE(S) Ability of macrophages to induce the adhesion phenotype in human peritoneal fibroblasts. RESULT(S) Hypoxia treatment resulted in a significant increase in TGF-β1 expression in human macrophages. Additionally, hypoxia treatment resulted in a significant increase in TGF-β1, VEGF, and type I collagen mRNA and protein levels in normal peritoneal fibroblasts compared with normoxic conditions. Similarly, normal peritoneal fibroblasts treated with media collected from macrophages cultured under hypoxic conditions resulted in a significant increase in TGF-β1, VEGF, and type I collagen mRNA and protein levels compared with normal peritoneal fibroblasts treated with media collected from macrophages cultured under normoxic conditions. Additionally, human peritoneal fibroblasts exposed to varying concentrations of TGF-β1 exhibited a dose-dependent response in the expression of TGF-β1, VEGF, and type I collagen. At a low TGF-β1 concentration (12.5 ng TGF-β1/mL medium), TGF-β1, VEGF, and type I collagen were significantly increased. In contrast, at higher TGF-β1 concentrations (25 and 50 ng TGF-β1/mL media), TGF-β1, VEGF, and type I collagen mRNA levels were significantly reduced compared with 12.5 ng TGF-β1/mL medium. CONCLUSION(S) Human macrophages, cultured under hypoxic conditions, release factors that induce the expression of the adhesion phenotype in normal peritoneal fibroblasts. Particularly, TGF-β1 reproduces this response by regulating the expression of TGF-β1, VEGF, and type I collagen in a dose-dependent manner. Therefore, these findings highlight an important role for human macrophages in peritoneal wound healing.

Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay

Abstract Sickle cell disease (SCD) is characterized by microvascular occlusion mediated by adhesive interactions of sickle erythrocytes (SSRBCs) to the endothelium. Most in vitro flow adhesion assays measure SSRBC adhesion during continuous flow, although in vivo SSRBC adhesive interactions occur during pulsatile flow. Using a well-plate microfluidic flow adhesion system, we demonstrate that isolated SSRBCs adhere to vascular cell adhesion molecule (VCAM-1) at greater levels during pulsatile versus continuous flow. A significant increase in adhesive interactions was observed between all pulse frequencies 1 Hz to 2 Hz (60–120 beats/min) when compared to non-pulsatile flow. Adhesion of isolated SSRBCs and whole blood during pulsatile flow was unaffected by protein kinase A (PKA) inhibition, and exposure of SSRBCs to pulsatile flow did not affect the intrinsic adhesive properties of SSRBCs. The cell type responsible for increased adhesion of whole blood varied from patient to patient. We conclude that low flow periods of the pulse cycle allow more adhesive interactions between sickle erythrocytes and VCAM-1, and sickle erythrocyte adhesion in the context of whole blood may better reflect physiologic cellular interactions. The microfluidic flow adhesion bioassay used in this study may have applications for clinical assessment of sickle erythrocyte adhesion during pulsatile flow.

A novel role of h2-calponin in regulating whole blood thrombosis and platelet adhesion during physiologic flow

Calponin is an actin filament‐associated protein reported in platelets, although the specific isoform expressed and functional role were not identified. The h2‐calponin isoform is expressed in myeloid‐derived peripheral blood monocytes, where it regulates adhesion. Our objective was to characterize the presence and function of the h2 isoform of calponin in platelets. H2‐calponin was detected in human and mouse platelets via Western blotting. Immunofluorescent staining demonstrated h2‐calponin and actin colocalized in both human and wild‐type mouse platelets at rest and following collagen activation. The kinetics of platelet adhesion and whole blood thrombosis during physiologic flow was evaluated in a microfluidic flow‐based thrombosis assay. The time to initiation of rapid platelet/thrombus accumulation (lag time) was significantly longer in h2‐calponin knockout versus wild‐type mouse blood (130.02 ± 3.74 sec and 72.95 ± 16.23 sec, respectively, P < 0.05). There was no significant difference in the rate of platelet/thrombus accumulation during the rapid phase or the maximum platelet/thrombus accumulation. H2‐calponin knockout mice also had prolonged bleeding time and blood loss. H2‐calponin in platelets facilitates early interactions between platelets and collagen during physiologic flow, but does not significantly affect the rate or magnitude of platelet/thrombus accumulation. H2‐calponin knockout mice take 2.3 times longer to achieve hemostasis compared to wild‐type controls in a tail bleeding model. The ability to delay platelet accumulation without inhibiting downstream thrombotic potential would be of significant therapeutic value, thus h2‐calponin may be a novel target for therapeutic platelet inhibition.

VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow

Very Late Antigen‐4 (VLA‐4, α4β1‐integrin, ITGA4) orchestrates cell‐cell and cell‐endothelium adhesion. Given the proposed role of VLA‐4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA‐4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA‐4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA‐4, while VLA‐4 staining of non‐SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA‐4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM‐1) was blocked by natalizumab in a dose‐dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF‐α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 μg/ml). This indicates that VLA‐4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM‐1 and that the VLA‐4 adhesion to VCAM‐1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA‐4 blockade may be beneficial in sickle cell disease.

Sevuparin blocks sickle blood cell adhesion and sickle-leucocyte rolling on immobilized L-selectin in a dose dependent manner

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TR2/TR4 overexpression in a humanized sickle cell disease mouse model decreases RBC adhesion to VCAM-1

Vaso-occlusion is a hallmark of sickle cell disease (SCD) accounting for much of its morbidity and mortality. The initiation and propagation of vaso-occlusion results from processes that impair blood flow through the microvasculature. In particular, sickle erythrocytes (SSRBCs) have a greater propensity to adhere to the vessel wall than non-sickle erythrocytes [1]. SSRBC adhesionmay directly occludemicrovessels or indirectly by delaying the transit time of SSRBCs through the capillary and propagating intra-capillary sickling. Very late antigen-4 (VLA-4) or α4β1 integrin is one of themost characterized RBC adhesionmolecules and supports adhesion between SSRBCs and endothelial vascular cell adhesion molecule 1 (VCAM-1) [2,3]. VLA-4 is expressed on immature RBCs/reticulocytes, and these cells are found in increased numbers in the peripheral blood of SCD patients [2,3]. Hydroxyurea (HU), a known inducer of fetal hemoglobin (HbF), is the only FDA-approved therapy for SCD and has been shown to improve morbidity and mortality in patients with SCD. There is increasing evidence suggesting that HU achieves this by decreasing the adhesive properties of SSRBCs to the endothelium and sub-endothelial matrix [4]. Not all patients respond to HU therapy, and HU is not well tolerated amongst all the responders. Thus, there is a need for alternative approaches to increase HbF levels to achieve similar or better clinical efficacy. Previous studies have shown that forced transgenic expression of TR2/TR4, a nuclear orphan hormone receptor that binds to the γglobin gene promoter, in sickle cell mice leads to induction of the γglobin gene [5]. Subsequent analysis revealed that elevated TR2/TR4 levels enhanced HbF production and improved the disease phenotype within SCDmice [5]. Thesefindings suggest that TR2/TR4may be a suitable therapeutic target to induce persistent HbF accumulation in patients with SCD. The current study was designed to determinewhether alleviation of the disease phenotype was due, in part, to a reduction in SSRBC adhesion to VCAM-1. A microfluidic-based flow systemwas utilized to measure erythrocyte adhesion to VCAM-1 during physiologic flow. Flow adhesion assays were performed with a flow system (Bioflux 1000Z; Fluxion, San Francisco, CA) utilizing the 48-well plate format. RBCs were perfused through VCAM-1 coated channels, as previously described [6]. SSRBCs from SCD:Tg mice demonstrated significantly lower adhesion to VCAM-1 when compared to SCD mice (69 ± 26 vs. 383 ± 80 RBCs/mm, respectively, p = 0.02). The normal reticulocyte fraction in blood is generally low, 0.5–1%. Chronic hemolytic anemia, a consequence of SCD, results in elevated numbers of circulating reticulocytes. VLA-4 is expressed on reticulocytes in high numbers. VLA-4 expression is usually lost from mature