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.

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.

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.

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)

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.

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.

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.

The iron chelator L1 potentiates oxidative DNA damage in iron-loaded liver cells.

Iron-mediated carcinogenesis is thought to occur through the generation of oxygen radicals. Iron chelators are used in attempts to prevent the long term consequences of iron overload. In particular, 1,2-dimethyl-3-hydroxypyrid-4-one (L1), has shown promise as an effective chelator. Using an established hepatocellular model of iron overload, we studied the generation of iron-catalyzed oxidative DNA damage and the influence of iron chelators, including L1, on such damage. Iron loading of HepG2 cells was found to greatly exacerbate hydrogen peroxide-mediated DNA damage. Desferrithiocin was protective against iron/hydrogen peroxide-induced DNA damage; deferoxamine had no effect. In contrast, L1 exposure markedly potentiated hydrogen peroxide-mediated oxidative DNA damage in iron-loaded liver cells. However, when exposure to L1 was maintained during incubation with hydrogen peroxide, L1 exerted a protective effect. We interpret this as indicating that L1's potential toxicity is highly dependent on the L1:iron ratio. In vitro studies examining iron-mediated ascorbate oxidation in the presence of L1 showed that an L1:iron ratio must be at least 3 to 1 for L1 to inhibit the generation of free radicals; at lower concentrations of L1 increased oxygen radical generation occurs. In the clinical setting, such potentiation of iron-catalyzed oxidative DNA damage at low L1:iron ratios may lead to long-term toxicities that might preclude administration of L1 as an iron chelator. Whether this implication in fact extends to the in vivo situation will have to be verified in animal studies.

Tissue factor expression by endothelial cells in sickle cell anemia.

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.

Circulating activated endothelial cells in sickle cell anemia.

BACKGROUND: The vascular wall participates in the pathogenesis of sickle cell disease. To determine whether the endothelium is activated in this disease, we studied the number, origin, and surface phenotype of circulating endothelial cells in patients with sickle cell anemia. METHODS: We used immunohistochemical examination of buffy-coat smears to enumerate circulating endothelial cells, and we evaluated the surface phenotype by applying preparations of circulating endothelial cells. An immunofluorescence microscopy panel of antibodies was used, including a specific anti-endothelial-cell antibody, P1H12. RESULTS: Mean (+/-SD) numbers of circulating endothelial cells in normal blood donors, patients with sickle cell trait, and patients with hemolytic anemias not due to hemoglobin S were 2.6+/-1.6, 3.0+/-2.6, and 2.0+/-0.8 per milliliter of whole blood, respectively. Patients with sickle cell anemia who presented with acute painful episodes had 22.8+/-18.2 circulating endothelial cells per milliliter of blood (P<0.001 for the comparison with normal donors), and patients with no such events within one month before or after blood sampling had 13.2+/-11.8 circulating endothelial cells per milliliter of blood (P=0.002 for the comparison with normal donors and P=0.019 for the comparison with patients with acute events). Serial observations of three patients showed a tendency toward higher levels of circulating endothelial cells at the onset of acute painful crises. The average viability of circulating endothelial cells was 66+/-30 percent. In patients with sickle cell anemia, regardless of clinical status, the circulating endothelial cells were predominantly microvascular in origin (CD36-positive), and most of the cells expressed four markers of endothelial-cell activation: intercellular adhesion molecule 1, vascular-cell adhesion molecule 1, E-selectin, and P-selectin. CONCLUSIONS: Our studies suggest that the vascular endothelium is activated in patients with sickle cell anemia, regardless of the patients' clinical status. Adhesion proteins on activated endothelial cells may have a role in the vascular pathology of sickle cell disease.

Removal of erythrocyte membrane iron in vivo ameliorates the pathobiology of murine thalassemia.

Abnormal deposits of free iron are found on the cytoplasmic surface of red blood cell (RBC) membranes in beta-thalassemia. To test the hypothesis that this is of importance to RBC pathobiology, we administered the iron chelator deferiprone (L1) intraperitoneally to beta-thalassemic mice for 4 wk and then studied RBC survival and membrane characteristics. L1 therapy decreased membrane free iron by 50% (P = 0.04) and concomitantly improved oxidation of membrane proteins (P = 0.007), the proportion of RBC gilded with immunoglobulin (P = 0.001), RBC potassium content (P < 0.001), and mean corpuscular volume (P < 0.001). Osmotic gradient ektacytometry confirmed a trend toward improvement of RBC hydration status. As determined by clearance of RBC biotinylated in vivo, RBC survival also was significantly improved in L1-treated mice compared with controls (P = 0.007). Thus, in vivo therapy with L1 removes pathologic free iron deposits from RBC membranes in murine thalassemia, and causes improvement in membrane function and RBC survival. This result provides in vivo confirmation that abnormal membrane free iron deposits contribute to the pathobiology of thalassemic RBC.

A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions: isolation by panning and stimulation with vascular endothelial growth factor.

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptor kdr, since mRNA for kdr was detected using RT-PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.

ETHICS AND STRATEGIC THERAPY: A PROPOSED ETHICAL DIRECTION.

Although ethical criticisms have been leveled against strategic therapy for some time, the current intensification of such criticisms indicates the need for continued dialogue about ethical strategic practice. This article presents ethical directions in two broad areas often seen as inherent in the strategic approach: (a) conscious deception and (b) intervention outside client awareness. Emerging from a discussion of the clinical implications of a constructivist view and a meaning system vocabulary of client change, five ethical presuppositions are exemplified. The ethical concerns of manipulation, power, and control are also discussed.

Strategic-brief therapy: an insight-oriented approach?*.

The role and importance of insight to the change process has been debated for many years. Strategists have given a significant amount of attention to discrediting insight and in the process have not considered its strategic advantages. This article will clarify the role of insight in strategic therapy and make distinctions between therapist-ascribed meaning (reframing) and interpretation, as well as client-ascribed meaning and insight. It will be argued that insight-oriented and strategic therapists share much in common and that insight, perhaps contrary to the current view, indeed has a role in the practice of strategic therapy.