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R P Hebbel

Publications and source records attributed to R P Hebbel.

At least 19 recordsLinked to original sources
PubMed2004

The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro.

When suspended in collagen gels, endothelial cells elongate and form capillary-like networks containing lumens. Human blood outgrowth endothelial cells (HBOEC) suspended in relatively rigid 3 mg/ml floating collagen gels, formed in vivo-like, thin, branched multi-cellular structures with small, thick-walled lumens, while human umbilical vein endothelial cells (HUVEC) formed fewer multi-cellular structures, had a spread appearance, and had larger lumens. HBOEC exert more traction on collagen gels than HUVEC as evidenced by greater contraction of floating gels. When the stiffness of floating gels was decreased by decreasing the collagen concentration from 3 to 1.5 mg/ml, HUVEC contracted gels more and formed thin, multi-cellular structures with small lumens, similar in appearance to HBOEC in floating 3 mg/ml gels. In contrast to floating gels, traction forces exerted by cells in mechanically constrained gels encounter considerable resistance. In constrained collagen gels (3 mg/ml), both cell types appeared spread, formed structures with fewer cells, had larger, thinner-walled lumens than in floating gels, and showed prominent actin stress fibers, not seen in floating gels. These results suggest that the relative magnitudes of cellular force generation and apparent matrix stiffness modulate capillary morphogenesis in vitro and that this balance may play a role in regulating angiogenesis in vivo.

Actins↗
PubMed2001

Modulation of endothelial cell activation in sickle cell disease: a pilot study.

The vessel wall endothelium undoubtedly plays a role in the vascular pathobiology of sickle cell disease. This pilot study tested the feasibility of using an inhibitor of nuclear factor (NF)-kappa B, a transcription factor, to modify the endothelial activation state of patients with this vascular disease. For a total of 7 separate drug exposure tests, 3 subjects with sickle cell disease took sulfasalazine (given orally at 1 g every 8 hours), and the activation state of their circulating endothelial cells (CECs) was assessed using immunofluorescence microscopy. Companion studies were also performed using sulfasalazine in sickle transgenic mice to verify its effect simultaneously on both CECs and vessel wall endothelium. Both CECs and tissue vessel wall endothelium in sickle mice have an activated phenotype. In these mice sulfasalazine significantly reduced CEC expression of vascular cell adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), and E-selectin, and it correspondingly reduced expression of these molecules in some tissue vessels. In humans with sickle cell disease, sulfasalazine significantly reduced CEC expression of VCAM, ICAM, and E-selectin, but it did not reduce expression of tissue factor. Addition of a second transcription factor inhibitor, salsalate, did not change this result. This pilot study suggests that endothelial cell activation state can be modified and down-regulated in vivo by sulfasalazine. (Blood. 2001;97:1937-1941)

Adult↗
PubMed2001

Oxidative stress and induction of heme oxygenase-1 in the kidney in sickle cell disease.

Chronic nephropathy is a recognized complication of sickle cell disease. Using a transgenic sickle mouse, we examined whether oxidative stress occurs in the sickle kidney, the origins and functional significance of such oxidant stress, and the expression of the oxidant-inducible, potentially protective gene, heme oxygenase-1 (HO-1); we also examined the expression of HO-1 in the kidney and in circulating endothelial cells in sickle patients. We demonstrate that this transgenic sickle mouse exhibits renal enlargement, medullary congestion, and a reduced plasma creatinine concentration. Oxidative stress is present in the kidney as indicated by increased amounts of lipid peroxidation; heme content is markedly increased in the kidney. Exacerbation of oxidative stress by inhibiting glutathione synthesis with buthionine-sulfoximine dramatically increased red blood cell sickling in the sickle kidney: in buthionine-sulfoximine-treated sickle mice, red blood cell sickling extended from the medulla into the cortical capillaries and glomeruli. HO activity is increased in the sickle mouse kidney, and is due to induction of HO-1. In the human sickle kidney, HO-1 is induced in renal tubules, interstitial cells, and in the vasculature. Expression of HO-1 is increased in circulating endothelial cells in patients with sickle cell disease. These results provide the novel demonstration that oxidative stress occurs in the sickle kidney, and that acute exacerbation of oxidative stress in the sickle mouse precipitates acute vaso-occlusive disease. Additionally, the oxidant-inducible, heme-degrading enzyme, HO-1, is induced regionally in the murine and human sickle kidney, and systemically, in circulating endothelial cells in sickle patients.

Anemia, Sickle Cell↗
PubMed2001

Identification and functional assessment of endothelial P1H12.

Monoclonal antibody P1H12 recognizes circulating endothelial cells and endothelia of all sizes of blood vessels. To identify the protein recognized by P1H12, we expressed a cDNA library in CHO cells and sequenced the cDNA from positive cells. The P1H12 sequence was identical, except at several bases, to that reported for melanoma cell surface antigen MUC18/CD146. Aggregation assays demonstrated that CD146 mediates Ca(++)-independent homotypic endothelial cell adhesion. P1H12 mAb abrogated interactions between human microvascular endothelial cells (HMVECs) but not between human umbilical vein endothelial cells (HUVECs). P1H12 mAb abrogated P1H12-positive (CHO(P1H12))-association with HMVECs or HUVECs. CD146 distribution is sparser on HUVECs than on HMVECs. These data imply that HMVECs and HUVECs express the CD146 binding partner but that CD146 is functional (or at sufficient density) only on HMVECs. HMVEC monolayers treated with soluble P1H12 mAb showed increased permeability to albumin, with accompanying changes in actin, paxillin, FAK, and caveolin distribution and changes in tyrosine phosphorylation of FAK. Stimulation with P1H12 mAb led to redistribution of NF-kappa B to the nucleus. P1H12 mAb bound to beads inhibited closure of wounded endothelial monolayers. CD146 thus joins VE-cadherin and PECAM-1 as a molecule that mediates homotypic endothelial cell adhesion. CD146 has both structural functions and signaling functions important for endothelial monolayer integrity.

Animals↗
PubMed2001

NHLBI workshop report: endothelial cell phenotypes in heart, lung, and blood diseases.

Endothelium critically regulates systemic and pulmonary vascular function, playing a central role in hemostasis, inflammation, vasoregulation, angiogenesis, and vascular growth. Indeed, the endothelium integrates signals originating in the circulation with those in the vessel wall to coordinate vascular function. This highly metabolic role differs significantly from the historic view of endothelium, in which it was considered to be merely an inert barrier. New lines of evidence may further change our understanding of endothelium, in regard to both its origin and function. Embryological studies suggest that the endothelium arises from different sites, including angiogenesis of endothelium from macrovascular segments and vasculogenesis of endothelium from microcirculatory segments. These findings suggest an inherent phenotypic distinction between endothelial populations based on their developmental origin. Similarly, diverse environmental cues influence endothelial cell phenotype, critical to not only normal function but also the function of a diseased vessel. Consequently, an improved understanding of site-specific endothelial cell function is essential, particularly with consideration to environmental stimuli present both in the healthy vessel and in development of vasculopathic disease states. The need to examine endothelial cell phenotypes in the context of vascular function served as the basis for a recent workshop sponsored by the National Heart, Lung, and Blood Institute (NHLBI). This report is a synopsis of pertinent topics that were discussed, and future goals and research opportunities identified by the participants of the workshop are presented.

Animals↗
PubMed2001

Whole blood tissue factor procoagulant activity remains detectable during severe aplasia following bone marrow and peripheral blood stem cell transplantation.

Using a novel whole blood assay, we recently demonstrated that tissue factor procoagulant activity (TF PCA) is present in normal individuals. Preliminary experiments suggested that this activity is localized in the mononuclear cell fraction. Postulating that whole blood TF PCA would therefore be undetectable when monocytes and neutrophils are absent from peripheral blood, we assayed TF PCA during the peri-transplant period in 15 consecutive patients undergoing allogeneic (n = 12) or autologous (n = 3) bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT). Baseline (pre-transplant) mean TF PCA was higher in patients compared to normal controls (P <0.005). Unexpectedly, although TF PCA during the period of profound aplasia was significantly reduced compared to baseline (p <0.05), fully 55% of the initial activity remained detectable. During the engraftment phase, TF PCA returned to pre-transplant levels, with a linear correlation between monocyte counts and TF PCA (r = 0.63). In contrast to normal whole blood, incubation of aplastic samples with E. Coli lipopolysaccharide ex vivo failed to induce TF PCA. Throughout the period of study--but especially during the aplastic phase--the absolute number of circulating endothelial cells (CECs) that were TF antigen-positive was increased compared to normals (P <0.001). However, removal of these cells from whole blood samples failed to significantly diminish total TF PCA indicating that CECs alone could not account for the detectable TF PCA during aplasia. We conclude that neither circulating mature myelo-monocytic cells nor endothelial cells can account for all the functionally intact TF in peripheral blood. Further studies are needed to identify the other source(s) of TF PCA.

Adolescent↗
PubMed2000

Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion.

Sickle cell anemia is characterized by painful vaso-occlusive crises. It is hypothesized that monocytes are activated in sickle cell disease and can enhance vaso-occlusion by activating endothelium. To test this hypothesis, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (MVEC) with sickle and normal mononuclear leukocytes were incubated, and endothelial activation was measured. Endothelial cells incubated with sickle mononuclear leukocytes were more activated than those incubated with normal mononuclear leukocytes, as judged by the increased endothelial expression of adhesion molecules and tissue factor and the adhesion of polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or platelets, were the mononuclear cells responsible for activating endothelial cells. Sickle monocytes triggered endothelial nuclear factor-kappa B (NF-kappaB) nuclear translocation. Cell-to-cell contact of monocytes and endothelium enhanced, but was not required for, activation. Antibodies to tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) blocked activation of the endothelium by monocytes. Peripheral blood monocytes from patients with sickle cell disease had 34% more IL-1beta (P =.002) and 139% more TNF-alpha (P =.002) per cell than normal monocytes. Sixty percent of sickle monocytes expressed the adhesion molecule ligand CD11b on their surfaces compared with only 20% of normal monocytes (P =.002). Serum C-reactive protein, a marker of systemic inflammation, was increased 12-fold in sickle serum than in normal serum (P =.003). These results demonstrate that sickle monocytes are activated and can, in turn, activate endothelial cells. It is speculated that vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of vaso-occlusion in sickle cell anemia.

Anemia, Sickle Cell↗
PubMed2000

Reperfusion injury pathophysiology in sickle transgenic mice.

Reperfusion of tissues after interruption of their vascular supply causes free-radical generation that leads to tissue damage, a scenario referred to as "reperfusion injury." Because sickle disease involves repeated transient ischemic episodes, we sought evidence for excessive free-radical generation in sickle transgenic mice. Compared with normal mice, sickle mice at ambient air had a higher ethane excretion (marker of lipid peroxidation) and greater conversion of salicylic acid to 2,3-dihydroxybenzoic acid (marker of hydroxyl radical generation). During hypoxia (11% O(2)), only sickle mice converted tissue xanthine dehydrogenase to oxidase. Only the sickle mice exhibited a further increase in ethane excretion during restitution of normal oxygen tension after 2 hours of hypoxia. Only the sickle mice showed abnormal activation of nuclear factor-kappaB after exposure to hypoxia-reoxygenation. Allopurinol, a potential therapeutic agent, decreased ethane excretion in the sickle mice. Thus, sickle transgenic mice exhibit biochemical footprints consistent with excessive free-radical generation even at ambient air and following a transient induction of enhanced sickling. We suggest that reperfusion injury physiology may contribute to the evolution of the chronic organ damage characteristic of sickle cell disease. If so, novel therapeutic approaches might be of value.

Allopurinol↗
PubMed2000

Desferrioxamine (DFO) conjugated with starch decreases NAD redox potential of intact red blood cells (RBC): evidence for DFO as an extracellular inducer of oxidant stress in RBC.

Desferrioxamine (DFO) is an important iron-chelating agent. It has also been thought of as an agent with anti-oxidant potential as it chelates ferric iron in various parts of the body. However, there is evidence suggesting that it may paradoxically affect red blood cells (RBCs) by inducing intracellular oxidant stress. Recently we observed that incubation of RBCs with DFO decreases NAD redox potential in normal RBC. To further understand the mechanism of DFO's interaction with RBC, we conducted a study to determine the effect of extracellular DFO upon RBC's redox status. We examined NAD redox potential in intact RBC (N = 7) incubated with DFO conjugated to starch. RBCs were incubated with 4 mM DFO for 3(1/2) hr and with 6 mM DFO for 2 and 3(1/2) hr. Significant decreases in NAD redox potential were observed after the incubations. With 4 mM DFO at the 3 (1/2) hr time point the mean decrease was 12.37% +/- 9.96% (P < 0.0085). With 6 mM DFO, the mean decreases were 18.54% +/- 9.79% (P < 0.0013) and 19.16% +/- 8.78% (P < 0.0006) for the 2 and 3 (1/2) hr incubations, respectively. DFO by itself is very poorly permeable to RBC. Conjugation with starch further ensured impermeability of DFO. The data presented here confirm the oxidant effect of DFO on RBC. The data also demonstrate that the effect of DFO on RBC's NAD redox potential originates extracellularly.

Adult↗
PubMed2000

Mechanisms of vascular instability in a transgenic mouse model of sickle cell disease.

We investigated a transgenic mouse model of sickle cell disease, homozygous for deletion of mouse beta-globin and containing transgenes for human beta(S) and beta(S-antilles) globins linked to the transgene for human alpha-globin. In these mice, basal cGMP production in aortic rings is increased, whereas relaxation to an endothelium-dependent vasodilator, A-23187, is impaired. In contrast, aortic expression of endothelial nitric oxide synthase (NOS) is unaltered in sickle mice, whereas expression of inducible NOS is not detected in either group; plasma nitrate/nitrite concentrations and NOS activity are similar in both groups. Increased cGMP may reflect the stimulatory effect of peroxides (an activator of guanylate cyclase), because lipid peroxidation is increased in aortae and in plasma in sickle mice. Despite increased vascular cGMP levels in sickle mice, conscious systolic blood pressure is comparable to that of aged-matched controls; sickle mice, however, evince a greater rise in systolic blood pressure in response to nitro-L-arginine methyl ester, an inhibitor of NOS. Systemic concentrations of the vasoconstrictive oxidative product 8-isoprostane are increased in sickle mice. We conclude that vascular responses are altered in this transgenic sickle mouse and are accompanied by increased lipid peroxidation and production of cGMP; we suggest that oxidant-inducible vasoconstrictor systems such as isoprostanes may oppose nitric oxide-dependent and nitric oxide-independent mechanisms of vasodilatation in this transgenic sickle mouse. Destabilization of the vasoactive balance in the sickle vasculature by clinically relevant states may predispose to vasoocclusive disease.

Anemia, Sickle Cell↗
PubMed2000

Origins of circulating endothelial cells and endothelial outgrowth from blood.

Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5-20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after approximately 1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only approximately 20-fold over this period, whereas donor-genotype endothelial cells expanded approximately 1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts.

Adult↗
PubMed2000

Hypoxia/reoxygenation causes inflammatory response in transgenic sickle mice but not in normal mice.

In sickle cell anemia, the initiation, progression, and resolution of a vasoocclusive episode may present features of ischemia-reperfusion injury, with recurrent episodes of ischemia/hypoxia and reoxygenation promoting inflammation. Here, we have tested the hypothesis that hypoxia/reoxygenation triggers inflammation in the transgenic sickle mouse. In these mice, even at ambient air, peripheral leukocyte counts are elevated by 1.7-fold and neutrophil counts by almost 3-fold. Two hours of hypoxia, followed by reoxygenation, induced a greater than normal rolling flux and adhesion of leukocytes in these mice, but no leukocyte extravasation. When 3 hours of hypoxia was followed by reoxygenation, sickle mice, but not normal mice, showed a distinct inflammatory response characterized by an increased number of adherent and emigrated leukocytes. Because these events, which are exaggerated in sickle mice, are not seen in response to hypoxia alone, we conclude that they represent a form of reperfusion injury. Studies using an H(2)O(2)-sensitive probe revealed clear evidence of oxidant production in vascular endothelial cells after hypoxia/reoxygenation in sickle mice. Infusion of an anti-P-selectin antibody, but not an anti-E-selectin antibody, completely inhibited this inflammatory response and significantly increased wall shear rates. These findings suggest that leukocyte-endothelium interaction contribute to vasoocclusive events in the sickle mice and perhaps in human sickle disease.

Anemia, Sickle Cell↗
PubMed1999

Sickle cell anemia as a possible state of enhanced anti-apoptotic tone: survival effect of vascular endothelial growth factor on circulating and unanchored endothelial cells.

The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% +/- 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% +/- 15% (P <.001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P =.001) higher in plasma of sickle donors (120.1 +/- 81.4 pg/mL) than that of normal donors (37.6 +/- 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r =. 612, P =.001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of alphavbeta3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.

Adult↗
PubMed1999

VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling.

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes endothelial cell survival and angiogenesis. We recently showed that VEGF can support the growth of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells in serum-free medium. Reasoning that VEGF might be modulating apoptotic signal transduction pathways, we examined mechanisms involved in the anti-apoptotic effect of VEGF on starvation- and ceramide-induced apoptosis in HDMEC. We observed that VEGF ameliorated the time-dependent increase in apoptosis, as demonstrated by morphologic observations, TUNEL assay, and DNA fragmentation. On the other hand, basic fibroblast growth factor only partially prevented apoptosis in serum-starved HDMEC; platelet-derived growth factor-BB was completely ineffective. VEGF activated the phosphorylation of extracellular signal regulated kinase (ERK)1 (p44 mitogen-activated protein kinase; MAPK) and ERK2 (p42 MAPK) in a time- and concentration-dependent manner. Both the VEGF-induced activation and its anti-apoptotic effect were prevented by the specific MAPK/ERK inhibitor PD98059. The presence of VEGF also inhibited the sustained activation of stress-activated protein kinase/c-jun-NH2-kinase (SAPK/JNK) caused by serum starvation and ceramide treatment. Activation of the MAPK pathway together with inhibition of SAPK/JNK activity by VEGF appears to be a key event in determining whether an endothelial cell survives or undergoes programmed cell death.

Apoptosis↗
PubMed1999

Mechanism of interaction of thrombospondin with human endothelium and inhibition of sickle erythrocyte adhesion to human endothelial cells by heparin.

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [125I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28-40%, (ii) the RGD peptide or MoAbs against integrin alpha v beta 3 or the calcium binding region of TSP inhibited binding by 18-28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.

Anemia, Sickle Cell↗