Nancy Gillett

An endothelial ligand for L-Selectin is a novel mucin-like molecule

The adhesive interaction between circulating lymphocytes and the high endothelial venules (HEV) of lymph nodes (LN) is mediated by lymphocyte L-selectin, a member of the selectin family of cell adhesion proteins. Previous work has identified a sulfated 50 kd glycoprotein (Sgp50) as an HEV ligand for L-selectin. We now report the purification of this glycoprotein and the utilization of the derived N-terminal amino acid sequence to clone a cDNA. The predicted sequence reveals a novel, mucin-like molecule containing two serine/threonine-rich domains. The mRNA encoding this glycoprotein is preferentially expressed in LN. Antibodies against predicted peptides immunoprecipitate Sgp50 and stain the apical surface of LN HEV. These results thus define a tissue-specific mucin-like endothelial glycoprotein that appears to function as a scaffold that presents carbohydrates to the L-selectin lectin domain.

Growth Hormone Improves Cardiac Performance in Experimental Heart Failure

BACKGROUND Growth hormone has been shown to increase maximum isometric active force of the left ventricular papillary muscle of rats in vitro. Administration of growth hormone causes an increase in myocardial contractility in normal humans. Our preliminary study suggests that treatment with growth hormone results in increased ventricular contractility in rats with left ventricular dysfunction. In the present study, the effects of growth hormone on cardiac function, including cardiac output, stroke volume, and peripheral vascular resistance, were determined in a rat model of heart failure. METHODS AND RESULTS Ligation of the left coronary artery or sham operation was performed; 4 weeks after surgery, recombinant human growth hormone (2 mg/kg per day SC) or vehicle then was administered for 15 days. The animals were catheterized after 13 days of the treatment. Cardiac output, measured by a thermodilution method, and other hemodynamic parameters were measured in the conscious animals 2 days after catheterization. The infarct sizes induced by left coronary ligation were comparable between growth hormone-treated and vehicle-treated rats. Six weeks after ligation, rats treated with vehicle exhibited significant decreases in cardiac index, stroke volume index, and left ventricular maximum dP/dt and increases in left ventricular end-diastolic pressure compared with sham rats. In the ligated rats, treatment with growth hormone increased cardiac index, stroke volume index, and left ventricular maximum dP/dt (P < .05) and reduced left ventricular end-diastolic pressure and systemic vascular resistance (P < .05). In sham rats, growth hormone slightly reduced arterial pressure but did not significantly alter cardiac performance. There was no significant difference in heart rate between the experimental groups. CONCLUSIONS These results suggest that growth hormone treatment may improve cardiac function by both increased myocardial contractility and decreased peripheral vascular resistance in heart failure.

Molecular cloning of a retina-specific membrane guanylyl cyclase

We have isolated and characterized cDNA clones encoding the human retinal guanylyl cyclase (retGC), a novel member of the membrane guanylyl cyclase gene family. Like other membrane guanylyl cyclases, the 1101 aa retGC is predicted to have a hydrophobic amino-terminal signal sequence followed by a large extracellular domain, a single membrane spanning domain, a kinase homology domain, and a guanylyl cyclase catalytic domain. In contrast to other membrane guanylyl cyclases, such as natriuretic peptide receptors, retGC has a relatively high basal level of activity when expressed in human 293 cells. cGMP production by retGC is unaffected by any of the known natriuretic peptides. In situ hybridization analysis of a variety of rhesus monkey tissues showed retGC transcripts to be localized exclusively along the retinal outer nuclear layer, corresponding to the nuclei of the rod and cone photoreceptor cells. Our results suggest that retGC may synthesize cGMP required for recovery of the dark state after phototransduction.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) mucin is expressed by lactating mammary gland epithelial cells and is present in milk.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) is a mucinlike endothelial glycoprotein that acts as an adhesive ligand for L selectin by presenting one or more O-linked carbohydrates to the lectin domain of this leukocyte cell surface selectin. The GlyCAM 1 glycoprotein has been previously shown to be expressed specifically by the endothelial cells of peripheral and mesenteric lymph nodes and in an unknown site in lung. Here we report that this protein is also expressed during lactation by mammary epithelial cells. Northern blot analysis has shown that the mRNA for GlyCAM 1 appears to be induced during pregnancy in a manner similar to that previously described for hormonally induced milk proteins. In situ hybridization analysis reveals that the site of GlyCAM 1 synthesis in the mammary gland is in the epithelial cells that produce these same milk proteins. Immunohistochemistry of mammary glands using antisera directed against GlyCAM 1 peptides demonstrates that these epithelial cells contain GlyCAM 1 protein, and that this protein is also found lumenally in the milk of the secreting mammary gland. Analysis of murine milk shows that immunoreactive GlyCAM 1 is found in the soluble whey fraction. Finally, labeling analysis of milk GlyCAM 1 has demonstrated that this form of the glycoprotein lacks the sulfate-modified carbohydrate that has recently been shown to be required for the ligand binding activity to L selectin. The nonsulfated mammary GlyCAM 1 is unable to interact with L selectin, consistent with the hypothesis that milk GlyCAM 1 has a different function than endothelial GlyCAM 1. These data thus suggest that milk GlyCAM 1 is a hormonally regulated milk protein that is part of the milk mucin complex. In addition, the finding that the mammary form of GlyCAM 1 contains different carbohydrate modifications than the endothelial form suggests that this glycoprotein may be a scaffold for carbohydrates that mediate functions in addition to cell adhesion.

Beneficial effects of growth hormone and insulin-like growth factor-1 in experimental heart failure in rats treated with chronic ACE inhibition

Summary: The effects of growth hormone (GH) plus insulin-like growth factor-1 (IGF-1) were tested in an experimental model of cardiac failure treated with chronic angiotensin-converting enzyme (ACE) inhibition. Myocardial infarction was induced in rats by left coronary artery ligation. Two weeks after ligation, the animals received either captopril (2 g/L in drinking water) or water for 3 months. The rats were then given either GH (2 mg/ kg/day) plus IGF-1 (2 mg/kg/day) or vehicle for 14 days. Captopril treatment decreased mean arterial pressure (MAP), left ventricular end-diastolic pressure (LVEDP) and systemic vascular resistance (SVR) (p < 0.05), and increased cardiac index (CI) and stroke volume index (SVI) (p < 0.05). GH/IGF-1 or captopril + GH/IGF-1 treatment decreased MAP, LVEDP, and SVR (p < 0.05), and increased left ventricular maximum dP/dt, CI, and SVI (p < 0.05). The increases in CI and SVI were significantly greater in the captopril + GH/IGF-1-treated animals than in those treated with captopril alone (p < 0.05). The beneficial effect of captopril in reducing cardiac hypertrophy was preserved in the captopril + GH/IGF-1 group. The results indicate that GH/IGF-l and captopril can improve cardiac performance in congestive heart failure by independent and complementary mechanisms.

An Optimized Protocol for In Situ Hybridization Using PCR-Generated 33P-Labeled Riboprobes

This report describes in detail an in situ hybridization protocol that has been optimized to produce consistent, sensitive results across a wide range of probe constructs and tissue types. Specific advances incorporated into this protocol include (1) the use of PCR gene fragments to generate cRNA probes, (2) the use of 33P-labeled probes and (3) the use of ultrafiltration microconcentrators to purify the probe and eliminate background. The use of 33PCR to generate RNA probes and the use of 33P-labeled probes in in situ hybridization have been described previously; however, this protocol is unique because it combines these techniques in a simple, streamlined procedure that includes the use of microconcentrators to purify the probe. This protocol is particularly appropriate for high-volume in situ hybridization laboratories using a wide variety of probe constructs and tissues.

Leukemia Inhibitory Factor Expression in Human Carotid Plaques: Possible Mechanism for Inhibition of Large Vessel Endothelial Regrowth

One common feature of atherosclerotic plaques is the denudation of the endothelium covering the plaque and subsequent failure of endothelial regrowth in contrast to a marked proliferation of neocapillaries arising from the vasa vasorum within the medial wall. Previous studies in vitro have demonstrated the ability of leukemia inhibitory factor (LIF) to potently inhibit aortic endothelial cell growth while only slightly inhibiting the growth of adrenal cortex capillary endothelial cells. This selective effect of LIF on endothelial cells from different sources suggests that it may play a role in the failure of endothelial regrowth in atherosclerosis. Sections of human carotid endarterectomy samples were examined by in situ hybridization for LIF mRNA expression. In one-third of the samples (4/12) examined, cells within the atherosclerotic plaque exhibited LIF expression. Immunohistochemistry of serial sections suggested that the LIF-positive cells were activated macrophages. These results suggest that LIF may play a role in the pathogenesis of atherosclerosis, particularly the denudation of the large vessel endothelium.

Effects of growth hormone in rats with postinfarction left ventricular dysfunction

SummaryGrowth hormone may affect cardiac function. In rats, chronic hypersecretion of growth hormone leads to increased maximum isometric contractile force of the left ventricular papillary muscle in vitro. In humans, administration of growth hormone can increase myocardial contractility. However, cardiac effects of growth hormone in heart failure or cardiac dysfunction have not been studied to date. The current study was to evaluate the cardiac effects of growth hormone in conscious rats with postinfarction left ventricular dysfunction and sham controls. Ligation of the left coronary artery or sham operation was performed, then 4 weeks after surgery, recombinant human growth hormone (2 mg/kg/day, SC) or vehicle was administered for 15 days. Catheters were implanted 13 days after treatment with growth hormone or vehicle. Hemodynamic parameters were measured in conscious rats 2 days after catheterization. In vehicle-treated rats, left ventricular systolic pressure, maximum dP/dt, and arterial pressure were significantly decreased and left ventricular end-diastolic pressure was significantly increased in the ligation group compared with sham controls. Growth hormone treatment increased left ventricular systolic pressure (p<0.05) and dP/dt (p<0.05) and reduced left ventricular end-diastolic pressure (p<0.05), significantly in the ligated rats. In sham rats, growth hormone tended to decrease arterial pressure but did not alter ventricular contractility. Neither ligation nor growth hormone significantly altered heart rate and right atrial pressure. These results suggest that growth hormone treatment may improve cardiac function by increasing myocardial contractility in cardiac dysfunction or heart failure.