The fibroblastic coconspirator in cancer progression.

A remarkable change has occurred in the thinking about epithelial-derived cancer in recent years: From almost entirely focusing on oncogenes and tumor suppressor genes has come the realization that the tumor microenvironment is a coconspirator in the carcinogenic process. Many types of stromal cells, including fibroblasts, adipocytes, macrophages, mast cells, and cells of the vascular system, are crucial contributors to epithelial carcinogenesis. Here, we focus on the fibroblast's role in cancer progression and the molecules involved in the communications between the fibroblasts and the cancer cells, including fibroblast secreted protein 1 (FSP-1 or S100A4), transforming growth factor beta (TGF-beta), the chemokine CXCL-12 (stromal derived factor 1 alpha, SDF-1alpha), type I collagen, and matrix metalloproteinase 13 (MMP-13).

Truncated ErbB2 receptor enhances ErbB1 signaling and induces reversible, ERK-independent loss of epithelial morphology.

Shedding of the extracellular domain of the ErbB2 tyrosine kinase receptor and expression of the remaining NH(2)-terminally truncated ErbB2 correlates with lymph node metastases and adverse outcome in human breast cancer. To study the possible signaling from such a truncated receptor, MCF-7 human breast cancer cells expressing NH(2)-terminally truncated ErbB2 (DeltaNErbB2) were compared with cells overexpressing wild-type ErbB2. Expression of DeltaNErbB2 in MCF-7 cells resulted in sustained activation of extracellular signal-regulated kinases (ERK) 1/2, extensive loss of the epithelial morphology, appearance of vesicles and long protrusions as well as pronounced scattering of the cells. Similar alterations were observed upon ErbB2 overexpression but at much lower levels. Employing cell clones with inducible expression of DeltaNErbB2, it was revealed that the morphological changes were fully reversible and depended on continuous expression of DeltaNErbB2 but not on the activation of the ERK1/2 pathway. Interestingly, the expression of DeltaNErbB2 resulted also in the increased expression and phosphorylation of ErbB1 as well as in the prolonged ligand-induced activation of the ErbB1 signaling pathway. In conclusion, constitutive signaling upon expression of the truncated ErbB2 receptor in human breast cancer cells promotes morphological changes indicative of a more motile and aggressive phenotype.

Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth.

Many solid tumors produce vascular endothelial growth factor C (VEGF-C), and its receptor, VEGFR-3, is expressed in tumor blood vessels. To study the role of VEGF-C in tumorigenesis, we implanted MCF-7 human breast carcinoma cells overexpressing recombinant VEGF-C orthotopically into severe combined immunodeficient mice. VEGF-C increased tumor growth, but unlike VEGF, it had little effect on tumor angiogenesis. Instead, VEGF-C strongly promoted the growth of tumor-associated lymphatic vessels, which in the tumor periphery were commonly infiltrated with the tumor cells. These effects of VEGF-C were inhibited by a soluble VEGFR-3 fusion protein. Our data suggest that VEGF-C facilitates tumor metastasis via the lymphatic vessels and that tumor spread can be inhibited by blocking the interaction between VEGF-C and its receptor.

BIBX1382BS, but not AG1478 or PD153035, inhibits the ErbB kinases at different concentrations in intact cells.

The activation of ErbB tyrosine kinase receptors (ErbB1, -2, -3, and -4) by ligand-induced homo- or heterodimerization regulates cell growth, death, and differentiation. AG1478 and PD153035 (also know as AG1517) have been adopted as specific ErbB1 inhibitors based on their high specificity for ErbB1 as compared to ErbB2 in in vitro kinase assays. We compared their ability to inhibit ErbB receptor signaling in intact cells to that of a novel ErbB receptor kinase inhibitor, BIBX1382BS. Neither AG1478 nor PD153035 displayed any specificity for ErbB1-mediated signaling induced by transforming growth factor alpha (TGF-alpha) as compared to signaling initiated through the other ErbB kinases. In contrast, BIBX1382BS was more potent at inhibiting signaling induced by TGF-alpha than that induced by neuregulin1-beta1 or anti-ErbB2 agonist antibodies. Interestingly, this compound blocked antibody-induced ErbB4 homodimer activation at even lower concentrations than ErbB1-triggered signaling. Thus, BIBX1382BS, but not AG1478 and PD153035, can be employed to differentiate between the ErbB kinases in intact cells when used at appropriate concentrations.

Cell death induced by TNF or serum starvation is independent of ErbB receptor signaling in MCF-7 breast carcinoma cells.

The ErbB receptor tyrosine kinase family consists of the epidermal growth factor (EGF) receptor (ErbB1) and three related receptors (ErbB2, ErbB3, ErbB4). Their intrinsic tyrosine kinases can be activated by receptor-dimerization induced by numerous ligands or overexpression. ErbB receptors are frequently overexpressed in breast cancer, and their overexpression is associated with protection from apoptosis. To directly assess their role in apoptosis sensitivity of breast cancer cells, we established MCF-7 breast carcinoma cell lines overexpressing each ErbB receptor alone or in all possible pairs. Overexpression of ErbB1, ErbB2 and ErbB4 receptors was enough to activate them as judged by their phosphorylation, whereas co-expression of other ErbB receptors was necessary for the phosphorylation of the ErbB3. Surprisingly, overexpression of the ErbB receptors even when combined with treatment with their ligands (EGF, transforming growth factor alpha, betacellulin, neuregulins) failed to protect the MCF-7 cells from cell death induced by either tumor necrosis factor (TNF) or serum starvation. During starvation TGF-alpha, however, increased the cell size of the ErbB1 overexpressing cell line, and neuregulin1-beta1 increased that of all cell lines. In conclusion, our data does not support the role of ErbB receptors in the regulation of cell death induced by TNF or serum starvation, and the observed association in breast cancer may be due to other concomitant changes.

Chest roentgenogram in the evaluation of heart defects in asymptomatic infants and children with a cardiac murmur: reproducibility and accuracy.

OBJECTIVES: To evaluate the reproducibility and the accuracy of pediatric radiologists' assessments of chest radiographs with respect to the presence or absence of heart defects in children with an asymptomatic heart murmur. DESIGN: Ninety-eight children, ages 1 month to 15 years (median, 30.1 months), referred for evaluation of a heart murmur were consecutively included. They all had a standard chest radiograph and a color Doppler echocardiograph (CDE) performed. Six specialists in pediatric radiology evaluated the chest radiographs independently on two occasions 6 months apart. The radiologists were asked to classify each set of films into one of two categories: heart disease or no heart disease. The outcome of the CDE was considered the definite diagnosis. kappa statistics were used to analyze the reproducibility of the radiologic assessments. Sensitivity, specificity, and the predictive value of a positive and a negative test were used for evaluation of the accuracy of the radiologic assessments. RESULTS: Mean intra- and interobserver kappa values were all <0.6, and the majority were <0.4. Mean sensitivity was 0.3 (range: 0.17-0.52), mean predictive value of a positive test was 0.4, implying that 60% of the positive assessments were falsely positive. Mean specificity was 0.86 (range: 0.75-0.93) and the mean predictive value of a negative test was 0.80 implying that 20% of the negative assessments were falsely negative. CONCLUSION: We found a low reproducibility, as well as a low accuracy, of the radiologic assessments of the chest radiographs of children with an asymptomatic heart murmur with respect to the presence or absence of heart disease. A false-positive radiologic assessment of the chest radiograph with respect to heart defects causes unnecessary anxiety and further examinations, whereas a false-negative assessment might result in omission of relevant investigations and proper identification of the heart defect. We cannot recommend the use of chest radiographs in the initial evaluation of the asymptomatic child with a heart murmur. If a heart defect cannot be excluded by clinical examination a CDE must be performed.

Acquired antiestrogen resistance in MCF-7 human breast cancer sublines is not accomplished by altered expression of receptors in the ErbB-family.

Development of acquired resistance against antiestrogen treatment is a serious problem in human breast cancer, and knowledge of alterations resulting in resistance is important for selection of further treatment. To mimic the clinical situation we have established a series of MCF-7 human breast cancer cell lines by long term treatment with the antiestrogens tamoxifen, ICI 164,384, and ICI 182,780. Common for these cell lines is a decreased expression of the estrogen receptor alpha (ER alpha). In human breast cancer, lack of response to endocrine therapy is often associated with decreased expression of the estrogen receptor and increased expression of epidermal growth factor receptor (EGFR) and/or HER-2/neu (ErbB-2). Our antiestrogen resistant cell lines did not express altered levels of EGFR, HER-2/neu, ErbB-3, or ErbB-4. Estrogen and antiestrogen regulation of HER-2/neu expression was essentially similar in parent and resistant MCF-7 cells. Treatment with antibodies to HER-2/neu (Herceptin) did not affect growth of MCF-7 cells or resistant cells, indicating that in this in vitro model system, acquired antiestrogen resistance does not emerge from activation of the HER-2/neu signaling pathway. In MCF-7 cells transfected with HER-2/neu and/or ErbB-3, overexpression alone did not result in resistance. However, addition of heregulinl-beta1 abolished the inhibitory activity of ICI 182,780 on both vector and HER-2/neu/ErbB-3 transfected MCF-7 cells, demonstrating that activation of the HER-2/neu receptor signaling pathway can override the growth inhibitory effect of ICI 182,780.

Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases.

The major heat shock protein, Hsp70, is an effective inhibitor of apoptosis. To study its mechanism of action, we created tumor cell lines with altered Hsp70 levels. The expression levels of Hsp70 in the cells obtained correlated well with their survival following treatments with tumor necrosis factor, staurosporine and doxorubicin. Surprisingly, the surviving Hsp70-expressing cells responded to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3-like proteases in a manner essentially similar to that of the dying cells with low Hsp70 levels. However, Hsp70 inhibited late caspase-dependent events such as activation of cytosolic phospholipase A2 and changes in nuclear morphology. Furthermore, Hsp70 conferred significant protection against cell death induced by enforced expression of caspase-3. Thus, Hsp70 rescues cells from apoptosis later in the death signaling pathway than any known anti-apoptotic protein, making it a tempting target for therapeutic interventions.

Effect of nitrendipine on renal function and on hormonal parameters after intravascular iopromide.

PURPOSE: To evaluate the effect of the low-molecular nonionic radiographic contrast agent iopromide (Ultravist) on renal function, vasoactive peptides (angiotensin II, aldosterone, arginine vasopressin, and atrial natriuretic factor (ANF)), and blood pressure, and to evaluate the influence of the calcium antagonist nitrendipine on these parameters. The findings were evaluated in a prospective double-blind and placebo-controlled randomized study. MATERIAL AND METHODS: Twenty-six patients undergoing routine aortofemoral arteriography for peripheral atherosclerotic disease were treated with nitrendipine tablets (10 mg) or placebo twice daily for a week. Angiography was performed on the fifth day of medication. Efficacy variables were determined on the day before and 2 days after arteriography. The glomerular filtration rate and renal plasma flow were measured by the constant infusion technique. Renal tubular function was estimated from the clearance of lithium. Hormones were measured by radioimmunoassays. RESULTS: Arteriography with iopromide did not change renal function. No differences between the nitrendipine and placebo groups were found in renal hemodynamics, tubular sodium handling, or blood pressure. Nitrendipine changed ANF (26.1%) compared to placebo (1.5%), whereas the other hormones were not affected. CONCLUSION: The use of iopromide for angiography did not affect renal function in normotensive patients with peripheral atherosclerotic disease. Short-term treatment with nitrendipine may lower the plasma levels of ANF but it had no effect on renal function or blood pressure. Treatment with calcium antagonists prior to arteriography with iopromide is not indicated in these patients.

Involvement of caspase-dependent activation of cytosolic phospholipase A2 in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF)-induced apoptosis is mediated by caspases, which are cysteine proteases related to interleukin 1beta-converting enzyme. We report here that TNF-induced activation of caspases results in the cleavage and activation of cytosolic phospholipase A2 (cPLA2) and that activated cPLA2 contributes to apoptosis. Inhibition of caspases by expression of a cowpox virus-derived inhibitor, CrmA, or by a specific tetrapeptide inhibitor of CPP32/caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited TNF-induced activation of cPLA2 and apoptosis. TNF-induced activation of cPLA2 was accompanied by a cleavage of the 100-kDa cPLA2 to a 70-kDa proteolytic fragment. This cleavage was inhibited by Ac-DEVD-CHO in a similar manner as that of poly(ADP)ribose polymerase, a known substrate of CPP32/caspase-3. Interestingly, specific inhibition of cPLA2 enzyme activity by arachidonyl trifluoromethylketone (AACOCF3) partially inhibited TNF-induced apoptosis without inhibition of caspase activity. Thus, our results suggest a novel caspase-dependent activation pathway for cPLA2 during apoptosis and identify cPLA2 as a mediator of TNF-induced cell death acting downstream of caspases.

[Local intra-arterial thrombolysis. Results in arterial thromboses and graft occlusions]. / Lokal intraarteriel trombolysebehandling. Resultater ved arterielle tromboser og graft-okklusioner.

The purpose of this study was to evaluate the efficacy of intraarterial low-dose recombinant tissue plasminogen activator (rt-PA) in patients with acute or subacute thrombosis of the native arterial system or in an arterial artificial or venous by-pass graft. Twenty patients with 22 cases of thrombosis received intraarterial thrombolysis during the period 1.1.1995 to 31.12.1995. Ten patients (50%) achieved complete radiological thrombolysis, and in seven patients (35%) partial thrombolysis was achieved. Half of these patients required a subsequent PTA or an operation. There were only three patients (15%) where no lysis occurred. The results seemed very satisfactory and we conclude that thrombolysis should be considered as the first-choice treatment in this category of patients. The indications may be extended to patients with critical ischaemia with the introduction of the pulse-spray technique as the time to complete lysis is considerably shorter with this technique.

[Chronic thromboembolic pulmonary hypertension]. / Kronisk tromboembolisk pulmonal hypertension.

Acute pulmonary hypertension has a high mortality at the onset. Patients surviving the first phase will usually recanalize the pulmonary arteries through intrinsic thrombolytic mechanisms and medical treatment. However, in some cases there is insufficient resolution of the emboli with subsequent thrombotic and fibrotic reorganization, leading to a worsening of the pulmonary obstruction. In the open pulmonary arteries the disease may lead to hypertrophy of the media and intimal proliferation, thus leading to a further increase in the pulmonary vascular resistance. This again leads to hypertrophy of the right ventricle and ultimately to right-sided heart failure. Untreated, chronic thromboembolic pulmonary hypertension has a five-year mortality approaching 100%, but extensive pulmonary thrombendarterectomy using extracorporeal circulation and deep hypothermia has been shown to lower the pulmonary vascular resistance and thereby improve the prognosis significantly. Operative treatment can now be offered in Denmark, and the purpose of this review is to draw attention to the disease, its symptoms, diagnosis and the surgical treatment.

[Thromboendarterectomy of chronic thromboembolic pulmonary hypertension]. / Trombendarterektomi af kronisk tromboembolisk pulmonal hypertension.

Chronic thromboembolic pulmonary hypertension has a five year survival rate of less than 10% in patients with a systolic pulmonary artery pressure of 50 mmHg with no convincing effect of medical treatment. The operative mortality from pulmonary thrombendarterectomy in specialised centres has been reduced to 9%, suggesting this treatment as being an option. The results from thrombendarterectomy of two Danish patients are reported. The first patient, a 34 year-old woman was operated at the centre in San Diego with the assistance of a Danish thoracic surgeon. The second, a 60 year-old man was operated at our institution by this surgeon. Following removal of sufficient amount of embolic masses and intimal tissue, the patients were discharged from hospital with a substantial improvement in their clinical status and near normalisation of pulmonary artery pressure, which remained at the latest follow-up (3 to 22 months).

Diagnosing renal artery stenosis: a comparison between conventional renography, captopril renography and ultrasound Doppler in a large consecutive series of patients with arterial hypertension.

The purpose of the study was to compare the positive and negative predictive values of conventional renography (Reno-A), captopril renography (Reno-B) and ultrasound Doppler (UD) with regard to the diagnosis renal artery stenosis. These three tests, and in addition a renal angiography, were performed in consecutively admitted patients with arterial hypertension, owing to either suspicion of renovascular hypertension or refractoriness to treatment. Patients with occlusion of a renal artery or a serum creatinine level higher than 300 mumol/l, or a previous investigation for renovascular hypertension at another hospital, were excluded from the analysis. The European Multicenter Study (EMS) criteria and local criteria for abnormal renography were compared. Of 131 patients, 28 had a renal artery stenosis (RAS) exceeding 50% reduction in diameter of the artery and 19 exceeding 70%. Using the EMS criteria for renography the predictive values of a negative test for a RAS more than 50% were 0.88 for Reno-A, 0.90 for Reno-B, 0.86 for changes from Reno-A to Reno-B, 0.92 for abnormalities either in Reno-A, Reno-B or changes from Reno-A to Reno-B, and 0.91 for UD. The corresponding values for a RAS more than 70% were 0.94, 0.97, 0.93, 0.98 and 0.96, respectively. The predictive values of a positive test were clearly lower, ranging from 0.20 to 0.75, but best when changes from Reno-A to Reno-B were used, 0.69-0.75. Using local criteria for renography the predictive values of a negative test were almost equal to those obtained by using the EMS criteria, but the predictive values of a positive test were slightly lower. It is concluded that conventional renography, captopril renography and ultrasound Doppler all are very good screening tests for renal artery stenosis, but the positive predictive values are clearly highest when using changes from conventional renography to captopril renography. It is suggested that captopril renography always should be performed when conventional renography is abnormal and vice versa to obtain the highest positive predictive value, on the assumption that total renal function is normal or almost normal, and that renal function is not absent in the affected kidney.

[Diagnosis of suspected thoracic aortic dissection. Focused on transesophageal echocardiography]. / Diagnostik ved mistaenkt torakal aortadissektion. Fokus på transøsofageal ekkokardiografi.

Over a five-year period (1990-1994), 72 consecutive patients were referred to transoesophageal echocardiography (TEE) on suspicion of thoracic aortic dissection. TEE was performed as the only or last investigation in 42 patients (58%). In 44 patients one or more other investigations were carried out before final clinical decision making: aortography (n = 30), X-ray computer tomography (CT, n = 18), and magnetic resonance imaging (MRI, n = 12). The final diagnosis was based on the combination of clinical information, the available examination results, and findings at surgery or autopsy; 31 of the patients were diagnosed as having aortic dissection. One patient with aortic dissection died during TEE while none of the other patients suffered major complications. The sensitivity (demonstration of dissection including correct classification in type A or B) was 81%, 80%, 45%, and 83% for TEE, aortography. CT, and MRI, respectively. The specificities were 88%, 93%, 71%, and 100%, respectively. Dissection of the thoracic aorta is a life-threatening condition demanding prompt and accurate diagnosis. None of the four techniques employed in the present study is ideal. Although TEE is adequate for immediate bedside examination our results show that more time-consuming and resource demanding investigations are sometimes required. Proper training and improved equipment may, however, increase the usefulness of TEE in patients with suspected aortic dissection.

Endothelin in renovascular and essential hypertension.

Immunoreactive endothelin (ir-ET) was measured in peripheral venous plasma in 12 patients with renovascular hypertension (RVH) due to unilateral renal arterial stenosis, in 12 patients with essential hypertension (EH), and in 12 control subjects (C). In the patients with RVH, ir-ET was also measured in the aorta and in both renal veins before and 1 h after 25 mg of captopril was given orally. In peripheral venous plasma, ir-ET was the same in RVH (median 1.02 pmol/l (range 0.53-1.65)) as in EH (0.96 pmol/l (0.76-1.32)) and in C (1.00 pmol/l (0.77-1.16)). In RVH, the concentrations of ir-ET decrease from the aorta to the renal vein of both the affected (0.88 pmol/l (0.54-1.28) vs 0.68 (0.51-1.24), p < 0.01) and in the unaffected kidney (0.85 pmol/l (0.62-1.38) vs 0.78 pmol/l (0.36-1.25), p < 0.01). Renal extraction of ir-ET was the same on the affected side (15.1% (-3.7-33.2)) and on the unaffected side (11.2% (0.5-46.4)). In the aorta, ir-ET was significantly lower than in peripheral venous plasma (p < 0.05). The renal handling of ir-ET did not change in response to captopril in either the affected or unaffected kidney. It is concluded that circulating levels of ir-ET are normal in renovascular hypertension associated with unilateral renal artery stenosis and in essential hypertension. There is significant renal extraction of ir-ET which is unaffected by renal artery stenosis and captopril.

Thrombosis and intrinsic fibrinolysis in percutaneous transluminal angioplasty.

Coagulation and fibrinolysis were investigated in 14 claudicants undergoing percutaneous transluminal angioplasty (PTA) for femoropopliteal artery lesions. Cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (t-PA) antigen, fibrinopeptide A (FPA), and plasminogen activator inhibitor-1 (PAI-1) activity were measured in peripheral blood. XL-FDP and t-PA increased, and FPA and PAI-1 decreased significantly after angioplasty. XL-FDP increased from baseline 266 +/- 72 ng/ml to 481 +/- 239 ng/ml (p < 0.0005) 30 min after PTA, indicating mural thrombus formation in spite of the significant fall in FPA influenced by heparin. A groin haematoma developed after PTA in 4/6 patients, who received more than 5600 IU heparin and in 1/8 patients receiving smaller dosages. The alterations in PAI-1 showed no correlation with those of t-PA, whereas heparin had a sparing effect on PAI-1 consumption. These findings may indicate that PAI-1 acts as a thrombin inhibitor following deep vessel wall injury by angioplasty. In two patients, who had signs of rethrombosis on the next day, residual FPA was relatively high, XL-FDP peaked at 3530 +/- 1170 ng/ml, and t-PA increased by 2.6 +/- 1.0 ng/ml. The corresponding values in patients with an uncomplicated course were 406 +/- 89 ng/ml (p < 0.0001) and 0.1 +/- 0.5 ng/ml (p < 0.02). We conclude that thrombin promotes activation of coagulation and fibrinolysis in femoropopliteal PTA. Instability between these counteracting systems resulting in thrombosis is not prevented by conventional heparin administration at dosages causing bleeding complications.

Effect of captopril on the renal veno-arterial gradient of erythropoietin and oxygen in unilateral renal artery disease.

Twenty-nine patients with unilateral renal artery stenosis or occlusion were investigated. The veno-arterial gradient (VA-gradient) of erythropoietin (EPO), haemoglobin oxygen saturation and plasma renin activity (PRA) was determined separately in each kidney before and 1 h after angiotensin converting enzyme inhibition (ACE-inhibition). The VA-gradient of EPO and of hemoglobin oxygen saturation were the same in the affected and unaffected kidney during basal conditions. During ACE-inhibition the VA-gradient of EPO disappeared on the affected side but not on the unaffected side. A fall in s-EPO after ACE inhibition was demonstrated in the renal vein on the affected side (-1.4 U l-1, p < 0.01), in the contralateral vein (-0.8 U l-1, p < 0.01) and in the aorta (-0.6 U l-1, p < 0.01). The O2-gradients were reduced on both sides after captopril, from 10.8-7.5% (p < 0.04) on the affected side and from 10.8-9.0% (p < 0.04) on the contralateral. It is suggested that the stimulated renin-angiotensin system may be important for EPO production in the affected kidney in unilateral renal disease.