Fred L. Minnear

Rearrangement of adherens junctions by transforming growth factor-β1: role of contraction

The signal transduction pathways that lead to disruption of pulmonary endothelial monolayer integrity by transforming growth factor-β1 (TGF-β1) have not been elucidated. The purpose of this investigation was to determine whether disassembly of the adherens junction is temporally associated with the TGF-β1-induced decrease in pulmonary endothelial monolayer integrity. Measurement of albumin clearance and electrical resistance showed that monolayer integrity started to decrease between 1 and 2 h post-TGF-β1 treatment and continued to slowly decrease over the next 6 h. Immunofluorescence microscopy of monolayers between 2 and 3 h post-TGF-β1 showed that β-catenin, plakoglobin, α-catenin, and cadherin-5 were colocalized both at the cell periphery and in newly formed bands that are perpendicular to the cell-cell border. At 4 h post-TGF-β1, cells began separating; however, β- and α-catenin, plakoglobin, and cadherin-5 could still be found at the cell periphery at areas of cell separation and in strands between separated cells. By 8 h, these junctional proteins were no longer present at the cell periphery at areas of cell separation. The myosin light chain kinase inhibitor KT-5926 prevented the TGF-β1-induced change in integrity but did not inhibit the formation of actin stress fibers or the formation of bands containing adherens junction proteins that were perpendicular to the cell-cell junction. Overall, these results suggest that adherens junction disassembly occurs after cell separation during TGF-β1-induced decreases in pulmonary endothelial monolayer integrity and that the loss of integrity may be due to the activation of a myosin light chain kinase-dependent signaling cascade.

Electrical impedance of cultured endothelium under fluid flow

AbstractThe morphological and functional status of organs, tissues, and cells can be assessed by evaluating their electrical impedance. Fluid shear stress regulates the morphology and function of endothelial cells in vitro. In this study, an electrical biosensor was used to investigate the dynamics of flow-induced alterations in endothelial cell morphology in vitro. Quantitative, real-time changes in the electrical impedance of endothelial monolayers were evaluated using a modified electric cell-substrate impedance sensing (ECIS) system. This ECIS/Flow system allows for a continuous evaluation of the cell monolayer impedance upon exposure to physiological fluid shear stress forces. Bovine aortic endothelial cells grown to confluence on thin film gold electrodes were exposed to fluid shear stress of 10 dynes/cm2 for a single uninterrupted 5 h time period or for two consecutive 30 min time periods separated by a 2 h no-flow interval. At the onset of flow, the monolayer electrical resistance sharply increased reaching 1.2 to 1.3 times the baseline in about 15 min followed by a sustained decrease in resistance to 1.1 and 0.85 times the baseline value after 30 min and 5 h of flow, respectively. The capacitance decreased at the onset of flow, started to recover after 15 min and after slightly overshooting the baseline values, decreased again with a prolonged exposure to flow. Measured changes in capacitance were in the order of 5% of the baseline values. The observed changes in endothelial impedance were reversible upon flow removal with a recovery rate that varied with the duration of the preceding flow exposure. These results demonstrate that the impedance of endothelial monolayers changes dynamically with flow indicating morphological and/or functional changes in the cell layer. This in vitro model system (ECIS/Flow) may be a very useful tool in the quantitative evaluation of flow-induced dynamic changes in cultured cells when used in conjunction with biological or biochemical assays able to determine the nature and mechanisms of the observed changes.

Priming of human monocyte superoxide production and arachidonic acid metabolism by adherence to collagen- and basement membrane-coated surfaces.

Monocytes (mφs) come into intimate contact with basement membranes and extracellular matrix proteins as they extravasate from the blood to the interstitium or to sites of tissue injury. We examined the in vitro effects of mφ adherence to an endothelial cell‐derived basement membrane or to purified extracellular matrix proteins on phorbol myristate acetate (PMA)‐stimulated superoxide production and prostanoid secretion. Elutriation‐purified human peripheral blood mφs were adhered to tissue culture wells that were precoated with the following purified proteins: bovine serum albumin (BSA), collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), or laminin (LM). To model the provisional matrix at sites of tissue injury, mφs were also adhered to wells coated with either denatured collagen type I or gelatin (GEL) or coated with basement membrane (BM) derived from endothelial cell monolayers. The mφs were adhered to the protein‐coated surfaces for 1 h at 37° in serum‐free medium and washed to remove nonadherent cells, and the number of adherent mφs was measured. Monolayers of mφs were also incubated for an additional 18 h, at which time both adherence and cell spreading were measured. PMA‐stimulated superoxide production by adherent mφs was determined after 1 and 18 h of adherence to the protein‐coated surfaces. PMA‐stimulated release of two prostanoids, prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), was measured after 18 h of mφ adherence to the surfaces. Following 18 h of adherence, PMA‐stimulated superoxide anion secretion and secretion of PGE2 and TxB2 were augmented or primed by mφs adherent to COL I, GEL, or BM. In contrast, no such priming effects were observed by mφs adherent to COL IV, FN, or LM. The results suggest that adherence to basement membranes, collagen type I‐containing surfaces in the interstitium, or denatured collagen at sites of tissue injury primes mφ respiratory burst and arachidonate metabolism to inflammatory agonists. Induction of priming events by substrate‐specific adherence may be an important factor regulating host defense functions of mφs in the extracellular matrix. J. Leukoc. Biol. 55: 423–429; 1994.

MECHANISMS OF NEUROGENIC PULMONARY EDEMA

The pulmonary edema that develops quickly after a variety of cerebral insults, such as head injury, epileptic seizures, intracranial hypertension, and subarachnoid hemorrhage, is often accompanied by rapid flooding of airways with a protein-rich edema fluid (Weisman, 1939; MacKay, 1950; Richards, 1963; Simmons et al., 1968; Ciongoli and Posner, 1972; Theodore and Robin, 1976; Fisher and AboulNasr, 1979; Bayne and Simon, 1981; Fein and Rackow, 1982; Lee and Kobrine, 1983). To lay the framework for an understanding of the genesis of neurogenic pulmonary edema, a schematic representation of the pulmonary capillary-tissue-lymphatic system is indicated in Figure 1. Transcapillary fluid filtration is governed by the four Starling forces represented in the equation (Fig. 1): capillary hydrostatic pressure (Pc), plasma colloid osmotic pressure (irp), interstitial hydrostatic pressure (Pi), and interstitial tissue colloid osmotic pressure (vi). The plasma protein concentration is greater than the tissue fluid and lymph protein concentrations (Fig. 1). The lymph flow represents an overflow system. The tight alveolar interepithelial junctions in the normal lung (Taylor, 1981) restrict solute and water movement into the airspaces (Fig. 1). The protein concentration of airway edema fluid in neurogenic pulmonary edema approached that of plasma (Theodore and Robin, 1976; Fein and Rackow, 1982), suggesting that increases in permeability of pulmonary capillaries and alveolar epithelium to proteins are prominently involved in the development of pulmonary edema. Morphological studies in experimental animal models have supported these clinical observations (Hucker et al., 1976; Minnear and Connell, 1981). Since pulmonary arterial hypertension has been observed with the pulmonary edema in experimental animals and patients after central nervous system (CNS) injury (Sarnoff and Sarnoff, 1952; Wray and Nicotra, 1978), a rise in Pc may also be an important factor in edema formation. A purpose of this review is to discuss the relative roles of increases in permeability vs. capillary hydrostatic pressure in the development of neurogenic pulmonary edema. The rise in intracranial pressure, which is often observed in conjunction with neurogenic pulmonary edema (Ducker and Simmons, 1968; Simmons et al., 1968), may be a requirement for the development of pulmonary edema. Intracranial hypertension elicits the cardiopulmonary responses such as systemic arterial hypertension, left atrial hypertension, and pulmonary vasoconstriction that may be involved in pulmonary edemagenesis. Therefore, in this review, these responses to intracranial hypertension, the mechanisms mediating these responses, and their role in the development of pulmonary edema will be discussed. Lesions of specific anatomical sites in the brainstem and hypothalamus have been shown to lead to pulmonary edema. The following section reviews the role of CNS structures in the genesis of neurogenic pulmonary edema.

STATIN DRUGS AND DIETARY ISOPRENOIDS AS ANTITHROMBOTIC AGENTS

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis. Additional processes may enhance nitric oxide production and induce other processes. Downregulation of thrombin-stimulated events should promote hypothrombotic or quiescent conditions that reduce cardiovascular disease, thus contributing to longevity.

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 1251‐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 1251‐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.

State-of-the-Art-Review : Statins Induce Hypothrombotic States?

Statins inhibit 3-hydroxy-3-methyl-glutaryl coenzyme A (HMGCoA) reductase, which synthesizes mevalonic acid in the isoprenoid pathways. These pathways lead to squalene and subsequently to cholesterol and related products (e.g., steroids, vitamin D, bile salts, lipopro teins) and have major branches producing cell regulatory substances (e.g., farnesyl- and geranylgeranyl conjugated proteins) (1,2). Although cholesterol reduc tion in blood has been widely believed to be beneficial (e.g., less available for accumulation by foam cells in atherosclerotic plaques), the ability of cholesterol reduc tion to mitigate the incidence and severity of cardiovas cular diseases has recently been questioned. Like others (3-10), we (11) believe that statins and other substances, for example, plant isoprenoids in the diet (12), have ben eficial antithrombotic properties arising through the in hibition of an isoprenoid product other than cholesterol. However, unlike others, we also believe that this isopren oid product has cell regulatory functions upregulated by thrombin stimulation. Moreover, through such cellular pathways, thrombin should upregulate its own genera tion, and statins and dietary isoprenoids should induce hypothrombotic states by downregulating thrombin gen eration (Fig. 1).

Protein, not adenosine or adenine nucleotides, mediates platelet decrease in endothelial permeability

Platelets and platelet-conditioned medium (PCM) decrease endothelial protein permeability in vitro. Adenosine and a > 100-kDa protein have previously been implicated as the soluble factors released from platelets that decrease endothelial permeability. The objective of this study was to further investigate the role of adenosine in this platelet response. Measurements of adenosine and its precursor adenine nucleotides by high-performance liquid chromatography were correlated with the assessment of permeability by 125I-labeled albumin clearance and electrical resistance across endothelial cell monolayers derived from the bovine pulmonary artery. PCM contained micromolar concentrations of AMP, ADP, and ATP, but adenosine was below detectable levels (< or = 0.1 microM). Adenosine deaminase, an enzyme that converts adenosine to inactive inosine, or an adenosine-receptor antagonist did not block the platelet- or PCM-mediated decrease in endothelial permeability. A < 3-kDa fraction of PCM that contained micromolar concentrations of AMP and ADP did not affect endothelial permeability, whereas a > 3-kDa fraction that contained much reduced levels of AMP and ADP significantly decreased permeability. This activity of PCM was sensitive to insoluble trypsin. This study rules out adenosine and adenine nucleotides as primary factors in the platelet-induced decrease in endothelial permeability and suggests that the active factor is a protein.Platelets and platelet-conditioned medium (PCM) decrease endothelial protein permeability in vitro. Adenosine and a >100-kDa protein have previously been implicated as the soluble factors released from platelets that decrease endothelial permeability. The objective of this study was to further investigate the role of adenosine in this platelet response. Measurements of adenosine and its precursor adenine nucleotides by high-performance liquid chromatography were correlated with the assessment of permeability by125I-labeled albumin clearance and electrical resistance across endothelial cell monolayers derived from the bovine pulmonary artery. PCM contained micromolar concentrations of AMP, ADP, and ATP, but adenosine was below detectable levels (≤0.1 μM). Adenosine deaminase, an enzyme that converts adenosine to inactive inosine, or an adenosine-receptor antagonist did not block the platelet- or PCM-mediated decrease in endothelial permeability. A <3-kDa fraction of PCM that contained micromolar concentrations of AMP and ADP did not affect endothelial permeability, whereas a >3-kDa fraction that contained much reduced levels of AMP and ADP significantly decreased permeability. This activity of PCM was sensitive to insoluble trypsin. This study rules out adenosine and adenine nucleotides as primary factors in the platelet-induced decrease in endothelial permeability and suggests that the active factor is a protein.

Platelet Phospholipids Decrease Vascular Endothelial Permeability via a Novel Signaling Pathway Independent of cAMP/Protein Kinase A

Maintenance of the vascular endothelium as a semipermeable membrane to the passage of water and protein affords protection against the development of tissue edema. Platelets contribute to this maintenance of the vascular endothelial barrier. A low platelet count in the blood causes purpuric hemorrhages in the skin1 and has been associated with an increase in vascular endothelial permeability to protein in various organ beds.2,3 Repletion with platelet-rich plasma reverses these changes.1–3 Platelet conditioned medium (PCM), which contains releasate from platelets, replicates the permeability-decreasing activity of whole platelets.4-7 Two important questions remain concerning platelets and endothelial permeability, that is, the identity of the platelet factor(s) and the cellular mechanism. Much effort has been focused on identifying the active platelet factor(s). Proposed mediators that have been eliminated from contention are serotonin, norepinephrine, cyclooxygenase metabolites, adenosine, adenine nucleotides, and a >100-kD protein. A recent publication by Alexander et al.4 suggested that the active platelet factor was lysophosphatidic acid (LPA). We used gel filtration chromatography to show that phospholipids attached to albumin are responsible for the decreased vascular endothelial permeability induced by PCM. Permeability was assessed across endothelial cell monolayers derived from bovine pulmonary artery using electric cell-substrate impedance sensing (ECIS), which measures real-time changes in endothelial electrical resistance. Activity was present in a Sephacryl S-200 fraction associated with the calibrated albumin standard. Subsequent studies demonstrated that activity could be found in the albumin immunoprecipitate of PCM (FIG. 1) and in the fraction of PCM bound to a Blue Sepharose column, implying that the active factor associates with albumin. To confirm that the activity of the albumin fraction that we obtained by immunoprecipitation was a phospholipid, the albumin immunoprecipitate was extracted with methanol. Activity was present in the methanol extract and removed from the extracted albumin immunoprecipitate (FIG. 1). Since LPA was proposed as the active

Fibronectin inhibition of platelet thrombus formation in an in vivo porcine model of vascular injury.

Platelets adhere and aggregate in response to exposed subendothelial matrix during vascular injury. The present study examines the effect of plasma fibronectin on platelet deposition at a site of vascular injury in an in vivo porcine model. The internal carotid arteries in anesthetized Yorkshire pigs were bilaterally exposed and the distal half of each vessel stripped of endothelium. Following stripping, one in situ carotid artery preparation was filled with 0.5 mg/ml porcine plasma fibronectin and the other artery filled with vehicle solution, to serve as a control. After five minutes, 6-7 x 10(9) 111Indium-labeled autologous platelets were infused via a femoral vein cannula, and carotid blood flow was re-established for 20 minutes. The vessel segments were excised and deposition of platelets determined. Vascular stripping increased platelet deposition 52-fold, as compared to unstripped vessel segments. Fibronectin pretreatment did not affect platelet deposition in control vessel segments but decreased platelet deposition by 77% in stripped vessel segments. Transmission and scanning electron microscopy indicated that reduced platelet deposition in the fibronectin treated group was due to decreased platelet aggregation rather than decreased adhesion.