Human homeobox HOXA7 regulates keratinocyte transglutaminase type 1 and inhibits differentiation.

Keratinocyte proliferation and differentiation result from expression of specific groups of genes regulated by unique combinations of transcription factors. To better understand these regulatory processes, we studied HOXA7 expression and its regulation of differentiation-specific keratinocyte genes. We isolated the homeobox transcription factor HOXA7 from keratinocytes through binding to a differentiation-dependent viral enhancer and analyzed its effect on endogenous differentiation-dependent genes, primarily transglutaminase 1. HOXA7 overexpression repressed transglutaminase 1-reporter activity. HOXA7 message markedly decreased, and transglutaminase RNA increased, upon phorbol ester-induced differentiation, in a protein kinase C-dependent manner. Overexpression of HOXA7 attenuated the transglutaminase 1 induction by phorbol ester, demonstrating that HOXA7 expression is inversely related to keratinocyte differentiation, and to transglutaminase 1 expression. Antisense HOXA7 expression activated transglutaminase 1, involucrin, and keratin 10 message and protein levels, demonstrating that endogenous HOXA7 down-regulates multiple differentiation-specific keratinocyte genes. In keeping with these observations, epidermal growth factor receptor activation stimulated HOXA7 expression. HOX genes function in groups, and we found that HOXA5 and HOXB7 were also down-regulated by phorbol ester. These results provide the first example of protein kinase C-mediated homeobox gene regulation in keratinocytes, and new evidence that HOXA7, potentially in conjunction with HOXA5 and HOXAB7, silences differentiation-specific genes during keratinocyte proliferation, that are then released from inhibition in response to differentiation signals.

Hepatic removal of 125I-DLT gelatin after burn injury: a model of soluble collagenous debris that interacts with plasma fibronectin.

The decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin-coated particles to simulate blood-borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 125I-labeled dilactitol tyramine (DLT-gelatin) as a model of soluble collagenous tissue debris. We studied its blood clearance as well as organ localization in normal and postburn rats. Fibronectin-deficient plasma harvested early after burn exhibited limited ability to support in vitro phagocytic uptake of the gelatinized microparticles by Kupffer cells in liver tissue from normal rats. However, Kupffer cells in liver tissue from normal and postburn rats phagocytized the test particles at a normal rate when incubated in normal plasma. The DLT-gelatin ligand bound to fibronectin in a dose-dependent manner as verified by its capture with anti-fibronectin coated plastic wells when coincubated with purified fibronectin. By gel filtration chromatography, the binding of fibronectin with the DLT-gelatin ligand was readily detected, resulting in the formation of a high-molecular-weight complex. In normal animals the plasma clearance and liver localization of 125I-DLT-gelatin was competitively inhibited by infusion of excess nonradioactive gelatin. The blood clearance and liver localization of the soluble gelatin ligand were also impaired after burn injury during periods of fibronectin deficiency similarly to the pattern observed with gelatin-coated microparticles. By autoradiography, the cellular site for the uptake of the 125I-DLT-gelatin was primarily but not exclusively hepatic Kupffer cells; 125I-DLT-asialofetuin and 125I-DLT-ovalbumin were removed by hepatocytes and sinusoidal endothelial cells, respectively. Thus, gelatin conjugated with 125I-DLT can be used to simulate blood-borne soluble collagenous tissue debris after burn. It rapidly binds to plasma fibronectin before its hepatic Kupffer cell removal, and its blood clearance is markedly delayed after burn injury during periods of plasma fibronectin deficiency.

Blood-borne fragments of fibronectin after thermal injury.

Fibronectin is an adhesive protein that can promote phagocytosis and endothelial cell adhesion. Plasma fibronectin declines following burn in animals and patients, potentially due to its complexing with circulating collagenous debris as well as its rapid binding to sites of tissue injury. Such depletion of fibronectin initiates an opsonic deficiency of the plasma. In view of the sensitivity of fibronectin to proteolytic enzymes, an additional factor that could contribute to the decrease of plasma opsonic activity after burn is the proteolytic fragmentation of fibronectin in the blood. In the current study, we determined if fibronectin fragments appear in the blood of anesthetized rats after a sublethal full-thickness skin burn of 15% to 16% of body surface. Plasma fibronectin concentration was quantified by enzyme-linked immunosorbent assay and the presence of fibronectin fragments in plasma was determined by immunoblot analysis. All blood was collected in an antiprotease mixture to yield final plasma concentrations of 0.15% EDTA, 3mmol/L phenylmethylsulfonyl fluoride, and 3 mmol/L iodoacetate to prevent degradation of fibronectin after sampling. Plasma fibronectin decreased 60% to 70% within 30 minutes post-burn, and this low level lasted for at least 4 hours. Within 30 minutes post-burn, two prominent fragments of fibronectin with a molecular weight of 110 +/- 2.2 kd and 122 +/- 3.3 Kd, respectively, were also detected in the plasma. Peak concentration of these fragments was detected at 60 minutes post-burn, but their level declined by 4 hours. By 4 hours, both bands appeared to resolve into doublets. To rule out the possibility that the fragments of fibronectin detected in the plasma were actually generated by coagulation enzymes activated at the site of peripheral blood sampling, rapid direct inferior vena cava sampling was performed, which also yield the presence of the fragments. Thus, fibronectin fragments exist in the plasma following thermal injury. Because fragments of fibronectin can compete with the intact fibronectin molecule with respect to its ability to stimulate macrophage phagocytosis, such fragments may contribute to altered systemic phagocytic host defense following thermal injury. Furthermore, because fibronectin peptides can compete with matrix fibronectin and impair adhesion of cultured endothelial cells, such circulating fragments may also influence the integrity of the vascular barrier.

Blood-borne collagenous debris complexes with plasma fibronectin after thermal injury.

Plasma fibronectin augments the clearance of blood-borne foreign and effete complexes by mononuclear phagocytes. The release of a "gelatin-like" ligand into plasma after thermal injury has been reported. We quantified the release of this collagenous debris from thermally injured skin, and its potential interaction with soluble fibronectin in plasma using anesthetized rats. Collagen-like material debris in the plasma was detected by assay of hydroxyproline. Fibronectin was measured by a double antibody enzyme-linked immunosorbent assay (ELISA) technique. Over a 24-hour postburn interval, plasma hydroxyproline increased from 6.7 +/- 0.6 micrograms/mL to a maximum of 19.0 +/- 3.3 micrograms/mL at 60 minutes postburn, and normalized by 6 hours. A direct correlation existed between the magnitude of burn injury and the increase in plasma hydroxyproline. In parallel, plasma fibronectin declined over a 15-minute to 2-hour period postburn, and normalized by 3 to 4 hours with rebound hyperfibronectinemia observed at 24 hours. The elevation in total plasma hydroxyproline was not due to an increase in plasma Clq (zero time, 26.2 +/- 1.4 micrograms/mL; 60 minutes, 23.9 +/- 1.1 micrograms/mL). Tracer studies with 125I-fibronectin showed that the acute decline of plasma fibronectin was due to its uptake by the liver and binding to sites of tissue injury. Total hydroxyproline in extracts of burn skin, used as an index of soluble collagenous material, rose from 15 +/- 3.3 micrograms/g skin at zero time to 129.3 +/- 43.7 micrograms/g skin by 5 minutes postburn, with a decline to 38 +/- 22 micrograms/g skin by 24 hours. The formation of circulating fibronectin-gelatin complexes in vivo was documented by cross-immunoelectrophoresis coupled with autoradiography using 125I-gelatin as a model ligand. Thus, collagenous tissue debris from burned skin may enter the plasma after thermal injury and directly complexes with soluble fibronectin before hepatic phagocytic clearance.

Perturbation of red blood cell flow in small tubes by white blood cells.

The flow of blood in the microcirculation is facilitated by the dynamic reduction in viscosity (Fahraeus-Lindquist effect) resulting from the axial flow of deforming erythrocytes (RBCs) and from the decrease in the ratio of cell to vessel diameter. RBC velocity exceeds that of average fluid velocity; however the slower moving white blood cells (WBC) perturb flow velocity and the ratio of cell to vessel diameter by obstructing red cell flow through formation of "trains" of red cells collecting behind the white cell. This effect of white cells was studied quantitatively in a model in vitro tubes less than 10 microns in diameter with the demonstration that flow resistance increases linearly with white cell numbers up to 1,000 WBC/mm3 at tube hematocrit of 17.7%. The increase in resistance exceeds the flow resistance of WBC and appears to relate directly to train formation. A mechanical model of train formation developed to predict WBC influence in flow resistance over the range of WBC studied reasonably fits observed WBC effects.

Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane.

Intracellular Ca2+ at concentrations ranging from 0 to 10 mumol/l increases the shear modulus of surface elasticity (mu) and the surface viscosity (eta) of human red blood cells by 20% and 70%, respectively. K+ selective channels in the red cell membrane become activated by Ca2+. The activation still occurs to the same extent when the membrane skeleton is degraded by incorporation of trypsin into resealed red cell ghosts, suggesting that the channel activation is not controlled by the proteins of the membrane skeleton and is independent of mu and eta. Incorporation of trypsin at concentrations ranging from 0 to 100 ng/ml into red cell ghosts leads to a graded digestion of spectrin, a cleavage of the band 3 protein and a release of the binding proteins ankyrin and band 4.1. These alterations are accompanied by an increase of the lateral mobility of the band 3 protein which, at 40 ng/ml trypsin, reaches a plateau value where the rate of lateral diffusion is enhanced by about two orders of magnitude above the rate measured in controls without trypsin. Proteolytic digestion by 10-20 ng/ml trypsin leads to a degradation of more than 40% of the spectrin and increases the rate of lateral diffusion to about 20-70% of the value observed at the plateau. Nevertheless, mu and eta remain virtually unaltered. However, the stability of the membrane is decreased to the point where a slight mechanical extension, or the shear produced by centrifugation results in disintegration and vesiculation, precluding measurements of eta and mu in ghosts treated with higher concentrations of trypsin. These findings indicate that alterations of the structural integrity of the membrane skeleton exert drastically different effects on mu and eta on the one hand and on the stability of the membrane on the other.

Erythrocyte-endothelial cell adherence in senescence and in hemolytic disorders.

Adherence of normal human erythrocytes to cultured endothelial cells when exposed to in vitro fluid shear forces typical of venules, capillaries and arterioles is very low, less than 0.03 percent, in the presence of an albumin-buffer but increases five fold by the effects of autologous plasma. Senescent erythrocytes obtained by density separation had an adherence frequency in the normal cell range, suggesting that predisposition to endothelial adherence is not a key factor in regulation of life span by the sequestration, identification and phagocytosis modulated by immune recognition. Sickle cells' frequency of adherence exceeds that of normal cells particularly at low shear forces calculated to resemble those of veins and sinuses, and increases five to ten fold when autologous plasma obtained during sickle crisis is present; these findings add support to the hypothesis that excessive tendency for sickle cell adherence may contribute to the vascular obstructive phenomena of the disorder. The behavior of hereditary spherocytes and thalassemic erythrocytes resembles normal cells, suggesting that hemolytic mechanisms are not significantly dependent on erythrocyte-endothelial cell interaction. Erythrocytes from patients having diabetes exhibit the characteristics of control cells, suggesting that their membrane adhesion to endothelium may not be critical to initiation of vessel wall pathophysiological processes; however, the behavior in relation to damaged endothelial cells may differ importantly.

Erythrocyte-endothelial cell adherence in sickle cell disorders.

Detachment of individual sickle erythrocytes from cultured endothelial cell monolayers has been evaluated by a fluid-shearing technique in an effort to quantitate adherence at shear forces that would be anticipated in the in vivo circulation. Nonirreversibly sickled cells (non-ISC) were more adherent at normal oxygen tensions than control cells. More than 1% non-ISC remained attached to the monolayer at forces greater than physiologic shear stresses in capillary and venous circulations, and many of the most avidly attached cells, once separated, immediately reattached to adjacent endothelial cells. These data suggest that hemoglobin S-containing erythrocytes may have a higher frequency of adherence in vivo in regions of low shear stress where prolonged erythrocyte-endothelial cell contact could occur. Some of these cells detached by shear force would subsequently reattach in in vivo conditions. Plasma-enhanced attachment frequency and plasma from blood in a case of sickle crisis caused further increase. These observations further support the concept that sickle erythrocyte-endothelial cell interaction may be a significant factor in initiation of vascular occlusive events in sickle cell disease.

Alterations by leukocytes of erythrocyte flow in microchannels.

The effects of leukocytes on blood rheology in the microcirculation may be predicted to result from the rheological characteristics of the individual white blood cell types and their behavior at entrances of small vessels; their influence on flow resistance of blood due to their disturbance of erythrocyte flow; and the obstruction caused by their adherence to endothelial cells or geometrical hindrance of their flow at irregular or narrow regions. Deformation of leukocytes in micropipettes indicates the relative rigidity of lymphocytes and blast cells from leukemias in contrast to granulocyte viscoelastic properties, and entry times for lymphocytes in 2.6-4.6-microns channels were 11-151 s but milliseconds in 9.1-micron tubes. Lymphocytes and erythrocytes rarely (less than 1%) adhered to cultured endothelium; however, granulocytes adhered avidly at shear stresses of 10-100 dyn/cm2, typical of microcirculation. In the 9.1-microns flow tube at Hct = 17.7 +/- 2.6%, increasing [WBC] caused a linear increase in flow resistance, but above [WBC] = 1000/mm3 resistance tended to plateau. These data support the interpretation that granulocyte adherence to endothelial cells with the potential for obstruction of flow may be a more significant rheological consequence of leukocytes in blood than their resistance to flow because of their relative rigidity or their influence on flow resistance by perturbation of the erythrocyte flow.

Influence of wall surface on the flow of blood through endothelial-lined glass tubes.

It has been proposed that the presence of endothelial cells lining small vessels decreases resistance to microcirculatory blood flow. Results of previous investigations have been inconclusive when fibrin-coated glass tubes were used to approximate the endothelial layer. Estimates of the decrease in apparent viscosity for blood flow in these tubes have ranged from near-zero to 50% when compared to flow in unlined glass tubes. The present study was devised to determine the effect of endothelial cell layer on blood flow in vessels under in vitro conditions in which pressure--flow relationships can be monitored precisely. Monolayers of human endothelial cells were grown over the major portion of the inner surface of glass tubes (I.D. = 1130 microns, length = 7.5 cm), and differential pressure and flow in the tubes were measured for plasma and suspensions of erythrocytes in plasma. Controls of phosphate buffer solution and culture medium were used to calculate inner diameters for both tube types. Results show statistically insignificant differences between apparent viscosities calculated from pressure-flow data in unlined and endothelial cell-lined tubes. These data indicate that the presence of endothelial lining does not have a marked influence on apparent viscosity of either blood or plasma when flowing through tubes of this diameter.

Blood viscosity and thrombosis: clinical considerations.

PIP: Thrombus formation depends on adherence of blood-formed elements to the intimal surface through platelet-vessel surface interaction, platelet release phenomena and aggregation, formation of fibrin, and the enmeshing of blood cells. Arterial thrombi involve platelet aggregation, whereas venous thrombi found in low flow or during stasis have greater proportions of erythrocytes and fibrin. It is not known if or how abnormalities of flow resistance, platelet thrombus formation, or endothelial and dynamic parameters affect the microcirculation, largely due to the difficulty of obtaining comprehensive data from these systems. Increases of fibrinogen observed in many disorders may result in minor changes in blood viscosity without known physiologic consequence, but in most disorders in which thrombosis is observed, the pathophysiologic mechanisms are multifactorial and abnormal blood viscosity is presumed to be a significant but not limiting component. Therapeutic approaches in thrombotic disorders should recognize which elements of the thrombotic triad predominate. In arterial disorders focus should be on platelet activity, and the objectives of venous thrombosis treatment include prevention of morbidity and death from pulmonary embolism, reduction of morbidity resulting from the acute thrombotic episode, and prevention of the postphlebitic syndrome. Pathology, mechanism, and treatment for specific thrombogenic disorders are described. Treatments suggested for hyperviscosity involve giving antibiotics during crises. Also discussed are thalassemia, paroxysomal nocturnal hemoglobinuria, polycythemia, cryoglobulinemia, paraproteinemia, diabetes mellitus, and disseminated intravascular coagulation. Studies have established a relationship between thromboembolic disease and oral contraceptives (OCs). The risk is only increased while the patient is taking OCs but is compounded in women undergoing surgery or who have a disorder which predisposes to venous disease. The risk for myocardial infarction or stroke is significantly increased when OCs are taken over age 35 and when there is hypertension, smoking, type-II hyperlipoproteinemia, and diabetes mellitus. The risk appears to be a function of estrogen dosage, causing a 25% mean increase in calf venous volume and 30% decrease in vein velocity of venous blood compared to controls. Low flow rates may contribute to venous thromboembolism. OCs may alter precisely regulated systems of coagulation and fibrinolysis and recent studies confirm abnormalities in the hemostatic system attributed to OCs. 16% of women taking OCs have a 60% or greater reduction in antithrombin III activity. The multiple effects of OCs often result in low-grade activation of the hemostatic system, potentially lowering the threshold to precipitate thrombus formation and possibly explaining the increased incidence of thromboembolic disease. Heparin appears to reverse many of these problems.^ieng

Electroviscous flow and electrophoretic motion during erythrocyte entry in a glass capillary in the presence of applied electric potential gradients.

One means of measuring an effect of erythrocyte deformability on red cell flow is to determine how long it takes erythrocytes to enter or flow through an aperture whose diameter is smaller than 8 micrometers. It is convenient to impose an electric potential difference across the aperture so that the passage of an erythrocyte can be monitored by electronic means. We report here that such electric potential gradients may have a large influence on red cell entry times, primarily through the mechanism of erythrocyte electrophoretic mobility.

Effects of the calcium-mediated enzymatic cross-linking of membrane proteins on cellular deformability.

Excess calcium binding affects the shape and dynamics of cellular deformation of human erythrocytes. It may be hypothesized that incorporation of calcium may modify cellular deformability by processes which include specific cross-linking of membrane proteins with resultant changes in cell shape and deformability. Since previous studies indicate that accumulation of calcium ions causes development of gamma-glutamyl-epsilon-lysine bridges in membrane proteins, under control of a membrane transamidating enzyme which specifically requires calcium ions for activation, experiments were devised to examine the relationship between cross-linking and deformability and to determine the effects of specific inhibitor of membrane protein cross-linking on the calcium-dependent modification of erythrocyte to the echinocytic shape. The elastic shear modulus of the membrane was not significantly affected by calcium-induced cross-linking, indicating that induced shape change, not altered elasticity, causes the observed reduction in cellular deformability. These findings support the interpretation that Ca++-induced and transamidase-catalyzed cross-linking of membrane proteins contributes to fixation of altered cellular shape and decreased cellular deformability.

Biochemical factors influencing erythrocyte deformability and capillary entrance phenomena.

Evaluation of the effects of biochemical modification of erythrocytes by low pressure filtration through Nuclepore filters of 2.8 and 3.0 micrometer pore diameter and glass micropipettes of 2.7 and 3.0 micrometer internal diameter and channel length greater than 100 micrometer indicates the relative sensitivity of the micropipette in detection of reduction of cellular deformability. In dynamic studies of erythrocyte entrance behaviour on micropipettes, the significance of induced relative sphericity on cellular deformability was evident. Reduction of intracellular ATP, cellular pH, sphere induction by bifunctional membrane protein cross-linking reagents and lead caused diminution in cellular deformability, attributable to the more spherical shape. Similarly echinocyte formation by effects of aged plasma, oleate and senscence, and stomatocyte induction by chlorpromazine caused marked decrease in capillary entry of cells having flow velocities of approximately 100 micrometer/s in contrast inhibitors of the glucose carrier, of calmodulin and manipulated decrease in membrane lipid of bilayer fluidity did not cause detectable change in cellular deformability. These data emphasize the relative importance of erythrocytic shape as a determinant of cellular deformability and suggest that necessity for adequate control of shape change is necessary when examining effects of biochemical manipulations on membrane elasticity and cellular deformability. The preliminary date from capillary entrance experiment indicate the importance of dynamic experiments in in vitro studies of microcirculation.

Abnormalities in the membrane material properties of hereditary spherocytes.

Mechanical measurements of intrinsic membrane material properties are used to characterize the defect in hereditary spherocyte membrane at a continuum level. The value of the surface elastic shear modulus is two-thirds as large as normal values, and the value of the yield shear resultant is one-third as large as normal values. The viscosity of the surface above the elastic-plastic transition appears normal. Under similar geometric conditions, the force required to fragment a hereditary spherocyte is about one-third as large as the force required to fragment a normal cell.

Parallel decrease of erythrocyte membrane deformability and spectrin solubility at low pH.

Reduction of pH over the range 6.0-4.5 results in a decrease of erythrocyte deformability in parallel with the induced progressive sphericity of cells. At low deformation rates employed, increase of hemoglobin viscosity was not significant. A decrease of membrane elasticity was detected in cells when sphering, the major determinant of cellular deformability, was prevented at pH 5.0 by hyperosmotic medium. The pronounced change of deformability and the reduced elasticity occurred at calculated intracellular pH values at which solubility in vitro of extracted erythrocyte spectrin is also markedly reduced. The parallel decrease of deformability and spectrin solubility supports the hypothesis that spectrin aggregation may contribute to regulation of erythrocyte deformability through effects on shape and membrane elastic characteristics.

Absence of one component of spectrin adenosine triphosphatase in hereditary spherocytosis.

The stimulation by calcium and magnesium of ATPase activity of isolated ghosts, of water-soluble protein (spectrin), and of residual vesicles, derived from normal erythrocytes and from hereditary spherocytes (H.S.), has been measured. The ATPase activity found in normal water-soluble protein (WSP) at low levels of calcium (0.1-2.0 mM) is essentially absent in H.S. water-soluble protein, but the ATPase activity with magnesium and with high levels of calcium (60-100 mM) is the same in H.S. and normal WSP. Compared to normal, H.S. ghosts have increased Mg2+-stimulated activity. This increased activity is retained by the sedimentable vesicles ("residue") after extraction of the ghosts with 0.025 mM EDTA. The Ca2+, Mg2+-ATPase associated with the calcium pump is not significantly different in H.S.