Adolf Grünert

Identification, culture, and characterization of pancreatic stellate cells in rats and humans☆☆☆

BACKGROUND & AIMS Until now, the basic matrix-producing cell type responsible for pancreas fibrosis has not been identified. In this report, retinoid-containing pancreatic stellate cells (PSCs) in rat and human pancreas are described, and morphological and biochemical similarities to hepatic stellate cells are shown. METHODS Electron and immunofluorescence microscopy (collagen types I and III, fibronectin, laminin, alpha-actin, and desmin) was performed using pancreatic tissue and cultured PSCs. Extracellular matrix synthesis was shown using quantitative immunoassay and Northern blot analysis. RESULTS PSCs are located in interlobular areas and in interacinar regions. Early primary cultured PSCs contain retinol and fatty acid retinyl-esters. Addition of retinol to passaged cells resulted in retinol uptake and esterification. During primary culture, the cells changed from a quiescent fat-storing phenotype to a highly synthetic myofibroblast-like cell expressing iso-alpha-smooth muscle actin (>90%) and desmin (20%-40%) and showing strong positive staining with antibodies to collagen types I and III, fibronectin, and laminin. As determined on protein and messenger RNA level, serum growth factors stimulated the synthesis of collagen type I and fibronectin. CONCLUSIONS The identification of PSCs, particularly in fibrotic areas, and the similarities of these cells to hepatic stellate cells suggest that PSCs participate in the development of pancreas fibrosis.

Platelet-Derived Growth Factors Stimulate Proliferation and Extracellular Matrix Synthesis of Pancreatic Stellate Cells: Implications in Pathogenesis of Pancreas Fibrosis

At present, the cell-cell interactions and molecular mechanisms of pancreas fibrogenesis are largely unknown. The purpose of this study was to investigate paracrine stimulatory loops between platelets and pancreatic stellate cells (PSC). Human PSC were obtained by outgrowth from fibrotic human pancreas. Native platelet lysate (nPL) and transiently acidified platelet lysate (aPL) were added to cultured PSC (passage 4 to 7) in the absence of serum. The synthesis of collagen types I and III and c-fibronectin (cFN) was demonstrated on protein (immunofluorescence and quantitative immunoassay) and mRNA (Northern blot) level. Using sections of human pancreas with acute pancreatitis, platelet aggregates in capillaries were demonstrated by transmission electron microscopy. nPL, and to an even greater extent aPL, significantly increased the synthesis of collagen types I and III and of c-FN (120 μl/ml aPL increased collagen type I concentration in PSC supernatants by 1.99 ± 0.17 times and c-FN of 2.49 ± 0.28 times, mean ± sd, n = 3). nPL and aPL also significantly stimulated cell proliferation (increased bromodeoxyuridine (BrdU) incorporation by 6.4 ± 0.78 times and 10 ± 0.29 times, respectively). By preincubating aPL with transforming growth factor β (TGFβ)- and platelet-derived growth factor (PDGF)-neutralizing antibodies and the TGFβ-latency associated peptide, respectively, TGFβ1 was identified as the main mediator stimulating matrix synthesis and PDGF as the responsible mitogen. Our data demonstrate that platelets contain fibrogenic mediators that stimulate proliferation (PDGF) and matrix synthesis (TGFβ1) of cultured PSC. We suggest that platelets and PSC cooperate in the development of pancreas fibrosis.

Effects of norepinephrine, epinephrine, and dopamine infusions on oxygen consumption in volunteers.

Objective:To determine the relationships between plasma concentrations of norepinephrine, epinephrine, and dopamine and oxygen consumption (Co2) during infusion of these catecholamines.Design:Prospective, randomized variable dose, pharmacologic study in which a noncumu-lative infusion-rate design wa

Plasma catecholamine concentrations after successful resuscitation in patients.

ObjectivesTo measure plasma catecholamine concentrations after cardiopulmonary resuscitation (CPR) and to correlate catecholamine concentrations with heart rate (HR), BP, and plasma glucose and lactate concentrations. DesignProspective, descriptive study. SettingEmergency medical service at a University Hospital. PatientsTen patients (58 to 85 yrs) with out-of-hospital cardiac arrest. InterventionsAt 1, 5, 15, 30, and 60 mins after restoration of spontaneous circulation, blood samples were drawn and BP and HR were measured. Plasma catecholamine concentrations were measured by high-pressure liquid chromatography and plasma glucose and lactate concentrations were measured by enzymatic methods. Main ResultsMedian plasma epinephrine and norepinephrine concentrations were 136.3 μg/L (136,300 pg/mL), range 27.6 to 397.6 μg/L (27,600 to 397,600 pg/mL), and 4.7 μg/L (4700 pg/mL), range 1.8 to 14.5 μg/L (1800 to 14,500 pg/mL), respectively, at 1 min. Median plasma epinephrine and norepinephrine concentrations decreased to 8.7 μg/L (8700 pg/mL), range 1.2 to 55.0 μg/L (1200 to 55,000 pg/mL) and 1.9 μg/L (1900 pg/mL), range 1.3 to 5.8 μg/L (1300 to 5800 pg/mL), respectively, at 60 mins after restoration of spontaneous circulation. Epinephrine concentrations decayed with a semilogarithmic decay pattern. The half-life for the phase was 2.2 mins and was 38.7 mins for the β phase. Mean values of systolic arterial pressure were between 136 ±PT 23 mm Hg at 1 min and 120 ±PT 15 mm Hg at 30 mins. Median plasma glucose concentrations were between 8.2 mmol/L (147.7 mg/dL; range 5.8 to 11.2 mmol/L [104.5 to 201.8 mg/dL]) at 1 min and 13.9 mmol/L (250.4 mg/dL; range 9.7 to 16.6 mmol/L [174.8 to 299.1 mg/dL]) at 30 mins. Lactate values were between 11.4 mmol/L (range 4.7 to 16.5) at 1 min and 5.2 mmol/L (range 2.7 to 12.5) at 60 mins. No significant correlations were found between circulating catecholamine concentrations and the other variables. ConclusionsAfter CPR, plasma catecholamine concentrations remained at high values but they did not lead to increases in BP, HR, or circulating glucose concentrations. (Crit Care Med 1992; 20:609–614)

Serum amyloid A versus C-reactive protein in acute pancreatitis: clinical value of an alternative acute-phase reactant.

Objectives: The acute‐phase reactant C‐reactive protein (CRP) is currently the serum variable of choice for an early, accurate, and cost‐effective severity assessment of acute pancreatitis in the daily clinical routine. Serum amyloid A (SAA) proteins comprise a family of apolipoproteins that constitute another major acute‐phase reactant and thus could be a potential alternative to CRP assessment. In the present study we investigated the clinical usefulness of SAA determinations in acute pancreatitis using an automated immunoassay technique. Design: Cohort study, comparing patients with complicated and mild acute pancreatitis; control groups included individuals with further abdominal disorders and healthy volunteers. Setting: A collaborative study between the department of general surgery and the routine laboratory of the department of clinical chemistry/pathobiochemistry. Patients: We enrolled 66 patients with acute pancreatitis in the present study. Control groups consisted of healthy subjects (n = 30), patients with chronic pancreatitis (n = 20), patients with pancreatic carcinoma (n = 20), and patients with acute appendicitis (n = 20). Interventions: Blood samples were collected during 14 consecutive days in patients with acute pancreatitis. A single blood specimen was taken in all control groups after the diagnosis was established. Measurements and Main Results: SAA concentrations were 3 mg/L (median; range, 3‐93) in healthy subjects. Although SAA and CRP both reached their maximum within 4 days after onset of symptoms in patients with acute pancreatitis, SAA concentrations rose faster above normal ranges and reached 676 mg/L (median; range, 12‐1880), higher than CRP, which reached 313 mg/L (median; range, 29‐613). As observed for CRP, SAA was significantly higher in patients who developed complications such as necrosis, infection of necrosis, or multiple organ dysfunction syndrome or in patients who died. SAA achieved best results in discriminating between necrotizing pancreatitis and interstitial edematous pancreatitis. However, CRP provided an earlier differentiation between both entities and a significantly better overall accuracy, as shown by receiver operating characteristics analysis. SAA concentrations in patients with chronic pancreatitis were 6 mg/L (median; range, 3‐756). In patients with pancreatic carcinoma, SAA concentrations were 7 mg/L (median; range, 3‐492), and in patients with acute appendicitis, they were 50 mg/L (median; range, 3‐2140). Conclusion: SAA is a nonspecific and rapidly produced variable in inflammatory abdominal disorders with a wider dynamic range than CRP. The current assay technique renders SAA an applicable and readily available variable under clinical routine conditions. In cases of acute pancreatitis, however, CRP is still superior to SAA for early and accurate stratification of patients with a complicated course.

Lipopolysaccharide-Activated Macrophages Stimulate the Synthesis of Collagen Type I and C-Fibronectin in Cultured Pancreatic Stellate Cells

We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421-435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 +/- 0.09-fold of control and c-fibronectin 1.89 +/- 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 +/- 0.2-fold and 2.80 +/- 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(alpha-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFbeta1/microgram of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFbeta1/microgram of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFbeta as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFbeta1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis.

Depending on their concentration oxidized low density lipoproteins stimulate extracellular matrix synthesis or induce apoptosis in human coronary artery smooth muscle cells.

Abstract Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 μg/ml oxLDL. Higher oxLDL concentrations (>25 μg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 μg/ml ascorbic acid, 25, 50 and 100 μg/ml oxLDL induced apoptosis within 6–8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

The Clinical Value of Procalcitonin in the Prediction of Infected Necrois in Acute Pancreatitis

ObjectiveInfection of pancreatic necrosis (IN) has a major impact on management and outcome in acute pancreatitis (AP). Currently, guided fine-needle aspiration (FNA) is the only means for an accurate diagnosis of IN. Procalcitonin (PCT), a 116 amino acid pro-peptide of calcitonin has been found in high concentrations in patients with sepsis. In the present study we analyzed the clinical value of serum PCT for predicting IN in AP and compared the results to guided FNA.DesignClinicaly study.SettingA collaborative study between the Departments of General Surgery and Clincal Chemistry/ Pathobiochemistry of the University of Ulm, Germany.Patients61 patients with AP entered this study and were stratified into three groups according to morphological and bacteriological data: I. 22 patients with edematous pancreatitis (AIP), II. 18 patients with sterile necrosis (SN), III. 21 patients with IN.Measurements and ResultsDuring an observation period of 14 days PCT was measured by immuonluminometry, CRP was determined by lasernephelometry on a routine base. In patients with IN overall PCT concentrations were significantly higher than in those with SN, whereas CRP levels did not differ in both groups. In contrast, only low concentrations of both parameters were found in patients with AIP. By ROC analysis the best PCT cut-off level for predicting IN or persisting pancreatic sepsis was obtained at ≥1.8 ng/ml. If this cut-off was reached on at least two consecutive days, IN could be predicted with a sensitivity of 95%, a specificity, of 88%, and an accuracy of 90%. Guided FNA achieved a sensitivity, specificity, and accuracy of 91%. 79%, and 84% in differentiating IN from SN, respectively. After surgical treatment of IN median PCT values continued to be significantly higher in patients with persisting pancreatic sepsis (n=12) compared to those with an uneventful postoperative course (n=7). Our results demonstrate that monitoring of serum PCT could serve as a noninvasive and accurate method to predict IN in AP as well as to select patients with persisting septic complications after surgical debridement

Serum half-life time determination of free and total prostate-specific antigen following radical prostatectomy—a critical assessment

OBJECTIVES All studies investigating the elimination kinetics of serum total (tPSA) and free (fPSA) prostate-specific antigen (PSA) were carried out in men undergoing radical prostatectomy. Radical prostatectomy itself could, however, have a major influence on the serum concentration of these tumor markers (e.g., perioperative fluid shift or blood loss). The purpose of our study was to determine the half-life time of fPSA and tPSA with special regard to the influence of the radical prostatectomy on the serum concentration of these tumor markers. METHODS Eleven men (mean age 63.2+/-7.2 years) with organ-confined prostate cancer who underwent radical prostatectomy were investigated (final pathologic Stage pT2pN0 or lower). Serum samples were obtained preoperatively and 0.25, 0.5, 1, 2, 4, 8, 12, 16, 24, 48, 72, 120, 168, and 240 hours after removal of the prostate. fPSA and tPSA and albumin and total protein serum concentrations were determined in all samples. RESULTS During the first 120 minutes after removal of the prostate, albumin and total protein serum concentrations continuously declined, with a half-life time of -104.5+/-28 minutes and -129.7+/-32 minutes, respectively. Serum decline of fPSA and tPSA followed a biphasic kinetic. During the initial alpha-phase, fPSA and tPSA serum concentrations decreased, with a half-life time of -69+/-10.3 minutes and -87.3+/-18.1 minutes, respectively. During the terminal beta-phase, the half-life time of fPSA and tPSA was -1152.2 minutes (0.8 days) and -3916.1 minutes (2.7 days), respectively. Between the alpha-phase half-life time of fPSA or tPSA and the half-life time of the total protein or albumin concentration decline, significant correlations were found. CONCLUSIONS These correlations indicate that the rapid decline of fPSA and tPSA directly after removal of the prostate (alpha-phase half-life time) is caused by the radical prostatectomy itself. The half-life time of the beta-phase reflects the biologic clearance of PSA. Therefore, the half-life time determination of PSA after radical prostatectomy is of limited value if the influence of the operation itself on the serum PSA concentration is not taken into account.

Increased endothelin release by cultured human smooth muscle cells from atherosclerotic coronary arteries

OBJECTIVES Endothelin, a 21-amino acid peptide initially purified from the medium of cultured endothelial cells, is a potent vasoconstrictor exerting its effects predominantly in a paracrine or autocrine manner. Recent data indicate that endothelin is also synthesized by cultured vascular smooth muscle cells and that endothelin is an effective stimulator of smooth muscle cell proliferation. This study aimed to investigate the endothelin release of cultured human smooth muscle cells, isolated from coronary plaques and from normal coronary tunica media, and to determine circulating endothelin concentrations in patients with coronary artery disease compared to control subjects. METHODS Coronary plaque material was extracted by thrombendarterectomy during aorto-coronary bypass grafting (n = 19). Segments of normal coronary arteries were obtained at autopsy (n = 33). Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells with antibodies against smooth muscle alpha-actin. Venous blood samples were drawn from patients with coronary artery disease undergoing cardiac catheterization (n = 32) and from control subjects (n = 38). Endothelin concentrations in culture medium and in plasma samples were measured by radioimmunoassay after Sep Pak C18 extraction. RESULTS Cultured smooth muscle cells, isolated from coronary plaques, released a significantly (P < 0.001) higher amount of immunoreactive endothelin into the culture medium (39.2 +/- 3.9 pg/10(4) cells, mean +/- s.e.m., 31 supernatant samples) than smooth muscle cells from normal coronary tunica media (3.9 +/- 0.8 pg/10(4) cells, 28 samples). Circulating endothelin concentrations were slightly elevated (P < 0.01) in patients with coronary artery disease (3.8 +/- 0.2 pg/ml) compared to control subjects (3.0 +/- 0.2 pg/ml). CONCLUSIONS These data suggest that the endothelin production is markedly increased in smooth muscle cells of coronary atherosclerotic plaques. The enhanced endothelin release may stimulate smooth muscle cell proliferation in a paracrine or autocrine manner and thus may contribute to the development or progression of coronary artery disease.