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The effects of intracoronary ultrapure human Interleukin-1 on the regional distribution of coronary blood flow (radioactive microspheres), contractile function (subendocardial ultrasonic length gauges) and systemic hemodynamics were studied in open-chest, anesthetized dogs (n = 7). Bolus doses of Interleukin-1 (10, 20, and 30 u) administered directly into the left anterior descending coronary artery increased coronary blood flow from 43 to 71, 80 and 87 ml/min, respectively. The increase in blood flow produced by Interleukin-1 was distributed uniformly to the subendocardium, midmyocardium, and subepicardium of the left ventricular free wall without effect on regional function or systemic hemodynamics. Indomethacin (1 mg/kg i.v.) attenuated the increase in blood flow, especially to the subepicardium. Due to the selective diminution of the Interleukin-1-mediated increase in subepicardial blood flow by indomethacin, the subendocardial to subepicardial perfusion ratio was increased by Interleukin-1 in the presence of indomethacin. The present results demonstrate that Interleukin-1 has direct coronary vasodilator actions, a portion of which is mediated by a product of cyclooxygenase metabolism.

To investigate cellular signaling mechanisms that influence chondrocyte production of inorganic pyrophosphate (PPi), which promotes calcium pyrophosphate dihydrate (CPPD) crystal deposition.Articular chondrocyte and cartilage cultures were stimulated with protein kinase C (PKC) activator and adenyl cyclase activator. Generation of extracellular PPi was measured.Adenyl cyclase activation resulted in diminished pyrophosphate generation. PKC activation stimulated pyrophosphate elaboration.Two signaling pathways, cAMP and PKC, modulate generation of extracellular pyrophosphate by cartilage and chondrocytes. They are novel targets for potentially diminishing extracellular pyrophosphate elaboration that leads to CPPD crystal deposition.

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. BCP crystals cause mitogenesis of articular cells and stimulate matrix metalloprotease production, thus promoting degradation of articular tissues. Previous work suggested that BCP crystal-induced cell activation required intracellular crystal dissolution, induction of proto-oncogene expression, and activation of signal transduction pathways involving protein kinase C and mitogen-activated protein kinases. Here we further elucidate the mechanisms of BCP crystal-induced cell activation as BCP crystals activate transcription factors nuclear factor kappaB and activator protein 1 in human fibroblasts. We confirm the role of protein kinase C in BCP crystal-induced mitogenesis in human fibroblasts. In contrast, we demonstrate that BCP crystals do not activate signal transduction pathways involving protein tyrosine kinases or phosphatidylinositol 3-kinase. These data further define the mechanism of cell activation by BCP crystals and confirm its selectivity, an observation that may have therapeutic implications.

Combined neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) inhibition produces greater acute hemodynamic effects than either treatment alone. We investigated whether BMS-182657 (BMS), which bears inhibitory activities against both NEP and ACE, elicited similar enhanced effects. BMS inhibited NEP and ACE, in vitro (IC50 = 6 and 12 nM, respectively) and the pressor response to Ang I in rats. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats sensitive to NEP inhibition but not to ACE inhibition, BMS at 100 mumol/kg i.v. lowered mean arterial pressure (MAP) from 180 +/- 6 to 151 +/- 5 mm Hg. In sodium-depleted, spontaneously hypertensive rats (SHR) sensitive to ACE inhibition but not to NEP inhibition, BMS at 100 mumol/kg p.o. lowered MAP from 151 +/- 4 to 123 +/- 5 mm Hg. Cardiomyopathic hamsters with heart failure were administered vehicle or one of the following (30 mumol/kg i.v.): the ACE inhibitor enalaprilat; the NEP inhibitor SQ-28603; or BMS. Enalaprilat and SQ-28603 had minimal hemodynamic effects. BMS decreased left ventricular end-diastolic pressure by 12 +/- 2 and 10 +/- 1 mm Hg and left ventricular systolic pressure by 27 +/- 2 and 23 +/- 3 mm Hg at 30 and 60 min, respectively (P.05 vs. each other group). These changes were associated with a 40% increase in cardiac output, a 47% decrease in peripheral vascular resistance and a lowering of MAP by 21 +/- 3 mm Hg at 60 min (P.05 vs. each other group). There were no significant differences in the changes in heart rate or left ventricular stroke work index among the four groups. Hence, BMS-182657 is a dual inhibitor of NEP and ACE, is antihypertensive irrespective of the activity of the renin-angiotensin system and has acute hemodynamic effects in hamsters with heart failure greater than those produced by selective inhibition of NEP or ACE. The NEP and ACE inhibitory activities of BMS-182657 act synergistically and mimic the interaction resulting from combining selective inhibitors of these enzymes.

A means to monitor intestinal allografts will be crucial for the future success of small bowel transplantation. We have previously demonstrated the ability of high-frequency ultrasound (US) to diagnose porcine intestinal ischemia in vitro. The aim of this study was to compare the histologic appearance of normal porcine small bowel versus bowel undergoing acute rejection, using 8.5-MHz US images. We allowed porcine heterotopic small bowel allograft transplants to reject and then removed, at scheduled intervals from postoperative day 0 to 12, specimens of both the transplanted bowel and the native bowel. We examined the tissues in vitro with an 8.5-MHz linear array US system then studied them histologically. Histologically, the earliest changes of rejection occurred at days 4 to 5, with mild submucosal edema, endotheliitis, and vasculitis affecting the small vessels; the mucosa remained normal. By days 7 to 8, the submucosal endotheliitis became more prominent, with focal thrombosed small vessels; the mucosa now appeared abnormal with flattened villi, erosions, and necrosis. By days 10 to 12, marked submucosal edema, vasculitis, endotheliitis, and necrotic mucosa were present. Ultrasonically, normal intestinal wall has five wall layers, corresponding to mucosa, submucosa, muscularis propria, and subserosal fat. The US criteria for abnormality were loss of folds, decreased numbers of echo layers, discontinuity of layers, and a homogeneous appearance. Using these US criteria, blinded observers differentiated normal from abnormal bowel wall after transplantation with a sensitivity of 84% and a specificity of 81%. Most of the errors occurred with the day 4 and 5 specimens, which appeared nearly normal on US. This study demonstrates that US can potentially differentiate normal from abnormal changes after small bowel transplantation in vitro, and may be useful in monitoring the transplanted intestine. Abnormal US findings might then warrant a biopsy procedure. Further studies are necessary to fully evaluate the potential clinical use of US in intestinal transplantation.

Endoscopic ultrasound imaging of the gastrointestinal wall can be performed through intraluminal fluid or by direct transducer contact with the wall. We tested the hypothesis that the ultrasound appearance of the gastrointestinal wall is influenced by the amount of pressure applied when the transducer is in contact with the tissue. Fresh autopsy specimens from the porcine gastrointestinal tract were examined in vitro using an 8.5-MHz linear array ultrasound system. As transducer pressure against the wall was increased from 0 to 10 KPa, changes were seen on the images in wall thickness, tissue echogenicity, and the number of layers. The stomach and rectum were more resistant to compression than the esophagus, duodenum, and colon. Wall echogenicity increased with increasing degrees of applied pressure and some layers were obliterated by this pressure. The second ultrasound layer, or deep mucosa, appeared to be the most susceptible to compression. Endoscopic ultrasound imaging artifacts should be reduced by limiting the amount of pressure applied to the wall with the transducer.

Cheung AHS, Wang KY, Jiranek GC, Odegaard S, Kimmey MB, Silverstein FE. Evaluation of a 20-MHz ultrasound transducer used in diagnosing porcine small bowel ischemia. The authors have previously demonstrated the ability of an 8.5-MHz linear array to detect moderate or severe intestinal ischemia in a porcine model. This study compares the ability of the 8.5-MHz linear array with a prototype miniature 20-MHz ultrasound (US) imaging probe in detecting small bowel ischemia. Five piglets were studied in which vascular clamps were applied to isolated jejunal pedicles, then released sequentially at hourly intervals to induce ischemia from 0 to 6 hours. After 24 hours of reperfusion, the tissue was removed and examined with both the 8.5-MHz linear array and the 20-MHz probe. A histologic examination also was done. The acoustical criteria used for interpretation were presence or absence of folds, number of echo layers, relative thickness of layers and homogeneity and continuity of layers. The 8.5-MHz system predicted the duration of ischemia with a kappa value of 0.66 ± 0.03, whereas the 20-MHz system had a kappa value of 0.49 ± 0.03. Both systems were able to distinguish normal or mild ischemia from moderate or severe ischemia with sensitivity and specificity rates of at least 94%. Both 8.5− and 20-MHz US systems detected intestinal ischemia in vitro. Further studies are indicated to determine the ideal frequency and design for a US system that can be used clinically.

To evaluate the use of ultrasound (US) to detect intestinal wall ischemia, we isolated segments of jejunum on single vascular pedicles in five piglets. We sequentially clamped these segments in intervals of 0 to 6 h, reperfused them for 24 h, and then examined them in vitro histologically and with an 8.5 MHz US scan. All segments were grossly viable except those with 6 h of ischemia. Histologically, mild submucosal edema developed after 1 to 2 h of ischemia; after 3 to 4 h, mucosal necrosis, loss of folds, worsening submucosal edema, and prominent neutrophilic infiltration occurred; after 5 to 6 h, severe mucosal necrosis with hemorrhage and submucosal edema was present. Ultrasonically, we saw five wall layers in the control group corresponding to mucosa, submucosa, muscularis propria, and subserosal fat. After mild (1 to 2 h) ischemia, all layers were present except for a discontinuity in layer 3. After moderate (3 to 4 h) ischemia, the five layers persisted but with a markedly thickened submucosal layer, reduced echogenicity, and flattened mucosal folds. With severe (5 to 6 h) ischemia, we observed a loss of all normal layers with no discernible architecture. Using these US criteria, blinded observers were able to differentiate normal/mild from moderate/severe ischemia with a sensitivity and specificity of 100%. These data suggest that US can differentiate, in vitro, normal from moderate and severe degrees of intestinal wall ischemia that correlates well with the histological appearance. Endoscopic US or surgically implantable US probes can potentially help diagnose clinical intestinal wall ischemia.

The disordered production of extracellular inorganic pyrophosphate (PPi) by cartilage contributes to calcium pyrophosphate dihydrate crystal formation and associated diseases. We have previously shown that a factor(s) derived from human platelets markedly stimulates the accumulation of PPi in the media of porcine articular cartilage in organ culture. This is the first known physiologic modifier of PPi production by cartilage. We report herein that platelet derived growth factor (PDGF) is not the platelet factor responsible for PPi stimulation and that the active factor is not mitogenic for chondrocytes. PDGF added to the media of articular cartilage explants in the presence of 0.5% (platelet-poor) plasma (PPP) produces 3.92 +/- 1.6 mumol/L PPi compared with 2.85 +/- 0.7 mumol/L PPi in cartilage exposed to PPP alone. The platelet extract (PE) and PDGF are mitogenic for adult articular chondrocytes in high-density monolayer cultures. Anti-PDGF antibodies block the mitogenic effects of PDGF and PE. The uptake of thymidine labeled with tritium is 157% of control in cells exposed to PE, PPP, and polyclonal goat immunoglobulin G (IgG); 114% of control in cells exposed to PE, PPP, and anti-PDGF antibody; 148% of control in cells treated with PDGF, PPP, and goat IgG; and 98% of control in cells treated with PDGF, PPP, and anti-PDGF antibody. However, anti-PDGF antibody has no effect on PPi accumulation. PPi levels are 17.22 +/- 1.6 mumol/L in media from cartilage treated with PPP, goat IgG, and PE and 17.62 +/- 2.2 mumol/L in cartilage exposed to PE, PPP, and anti-PDGF antibody. We have further characterized the platelet factor responsible for the stimulation of PPi by cartilage. It is not mitogenic for chondrocytes, and it is not PDGF.

Captopril is a remarkably effective new antihypertensive drug designed and developed as a potent and specific inhibitor of angiotensin-converting enzyme, a zinc metallopeptidase that participates in the synthesis of a hypertensive peptide, angiotensin II, and in the degradation of a hypotensive peptide, bradykinin. Earlier studies with a snake venom peptide (teprotride or SQ 20881) that could be administered only by injection demonstrated that specific inhibitors of angiotensin-converting enzyme could be highly effective as antihypertensive drugs, and helped to clarify the specificity and mechanism of action of the enzyme. A hypothetical model of the active center of angiotensin-converting enzyme based on its presumed analogy to the well characterized zinc metallopeptidase carboxypeptidase A was used to guide logical sequential improvements of a weakly active prototype inhibitor that led eventually to the highly optimized structure of captopril. The hypothetical working model of the active site of angiotensin-converting enzyme used to develop captopril continues to provide a firm basis for development of new types of specific inhibitors of this biologically important enzyme.

Synovial tissue hyperplasia in basic calcium phosphate deposition disease has been suggested to develop through the stimulation of cell growth by basic calcium phosphate (BCP) crystals deposited in joints. These crystals have been used in vitro to stimulate DNA synthesis in quiescent fibroblasts to experimentally study this proliferative disease. The stimulation of DNA synthesis, in density-arrested Balb/c 3T3 cells, by BCP crystals was inhibited after down-regulating protein kinase C activity with 12-O-tetradecanoyl-phorbol 13-acetate (TPA). No effect on platelet-derived growth factor (PDGF)-stimulated DNA synthesis was observed under the same conditions. The expression of c-myc and c-fos increased in response to BCP stimulation in a manner similar to the increase produced by stimulation with PDGF. The BCP stimulation of c-fos and c-myc messages was inhibited 60 and 90%, respectively, in TPA-pretreated, protein kinase C-down-regulated cells. The induction of these transcripts by PDGF was unaffected in cells pretreated with TPA. TPA was unable to stimulate c-fos and c-myc expression or DNA synthesis following protein kinase C down-regulation. Both PDGF and TPA stimulated phosphorylation of an 80-kDa protein, whereas BCP crystals had no effect on phosphorylation of this protein. The exposure of density-arrested Balb/c 3T3 cells to BCP crystals had no effect on high affinity epidermal growth factor receptor binding under conditions in which PDGF and TPA reduced epidermal growth factor binding. The data suggest that PDGF can act to stimulate c-fos and c-myc expression as well as DNA synthesis through a protein kinase C-independent pathway, whereas BCP crystals require at least endogenous levels of protein kinase C to stimulate these events.

From available clinical, radiographic, and synovial fluid findings, coupled with in vivo radiolabelled crystal turnover data and in vitro experimental data, a hypothesis has been formulated relative to the pathogenesis of BCP crystal deposition diseases (Fig. 2). Synovial lining cells phagocytose BCP crystals and particulate collagens in the joint fluid. During and/or after internalization these cells are stimulated in a variety of ways: 1) protease synthesis and secretion is relentlessly stimulated, which may damage joint tissues producing clinically evident loss of collagenous tissues including cartilage, bone, and tendon, and which may release additional amounts of crystals and particulate collagens into the synovial fluid, completing a vicious cycle; 2) PGE2 production is greatly augmented; 3) DNA synthesis is stimulated as a result of increased inositol phospholipid turnover and intracellular crystal dissolution. The increased number of synovial cells also augments the total local generation of proteases and prostenoids. Mechanical factors such as trauma or joint overuse also contribute to the pathogenesis of joint destruction as discussed in the article on the clinical aspects of BCP crystal deposition.

An in vitro model of ADA deficiency is selectively toxic to cartilage from immature rabbits with a greater effect on growth plate than articular cartilage. The selective toxicity observed appears to be the consequence of ATP depletion. These results support the hypothesis that the chondro-osseous dysplasia observed in patients with ADA deficiency is caused by the disordered metabolism that results from the enzyme deficiency.

To examine the repair collagens produced by cells in injured cartilage, the femoral articular surfaces of three groups of New Zealand white rabbits were injured by making both superficial and deep lacerations and drill holes. Eight weeks after surgery, the rabbits were killed and slices of injured articular cartilage were harvested. The types of collagen being synthesized at the site of these lesions were identified by labeling the recovered specimens in vitro with 3H-proline and by characterizing the collagen using sodium dodecyl sulphate electrophoresis, carboxymethyl cellulose chromatography, and cyanogen bromide peptide analysis. In all cases, tissue-specific type II ([alpha1 (II)]3) cartilage collagen was synthesized. Histological examination using toluidine blue showed that the chondrocytes bordering the cartilage defect produced by deep lacerations and drill holes responded by increased cellular activity, as shown by cell cloning and increased matrix staining. The drilled holes were completely filled by tissue with staining and morphological characteristics similar to those of hyaline cartilage.

The healing of surgically induced defects in mature rabbits' hyaline cartilage was examined histologically and biochemically. The subchondral bone underneath the lesion was the source of repair. At the end of the first month, mushroom-shaped chondroid buds were seen sprouting from the subchondral bone. By the tenth week, these buds fused to a cartilagenous plug filling the lesion. The repaired cartilage synthesized Type II collagen.

Collagenase has been implicated in the pathogenesis of several arthropathies. Arteparon [glycos-aminoglycan (GAG) polysulfate] is a proteinase inhibitor that is being investigated as a therapeutic agent in osteoarthritis. We found that cultures of calcified lapine synovium release collagenase, one tenth of which is already activated. Normal synovium produces no active collagenase. GAG polysulfate suppresses the amount of active collagenase by one half. GAG polysulfate may interfere with the activation process of collagenase.

Nachweis mit Analogen der im Gifte vonBothrops jararaca gefundenen Peptide, dass die Inhihibition des Angiotensins «converting enzyme» von zwei eindeutigen Teilsequenzen dieser Peptide abhangig ist. Die hohe spezifische kompetitive Inhibition, hervorgerufen durch die Peptide vonBothrops jararaca, wird der Bindung ihrer Tripeptidreste vom Carboxyterminus mit dem aktiven Zentrum des Enzyms zugeschrieben, die in gleicher Weise wie die Peptidsubstrate mit dem Enzym gebunden werden. Die Wirksamkeit der Giftpeptide hangt von der Bindung eines zweiten Teiles der Peptide mit dem Enzym ab.