Chronic treatment with a broad-spectrum metalloproteinase inhibitor, doxycycline, prevents the development of spontaneous aortic lesions in a mouse model of vascular Ehlers-Danlos syndrome.

There is no proven therapy or prevention for vascular Ehlers-Danlos syndrome (vEDS), a genetic disorder associated with the mutation of procollagen type III and characterized by increased fragility of vascular and hollow organ walls. Heterozygous COL3A1-deficient (HT) mice recapitulate a mild presentation of one of the variants of vEDS: haploinsufficiency for collagen III. Adult HT mice are characterized by increased metalloproteinase (MMP) activity, reduced collagen content in the arterial walls, and spontaneous development of various severity lesions in aorta. We hypothesized that chronic treatment with a MMP inhibitor would increase collagen content and prevent the development of spontaneous aortic lesions. HT mice were treated since weaning with the broad-spectrum MMP inhibitor doxycycline added to food. At the age of 9 months MMP-9 expression was twice as high in the tunica media of aorta in untreated HT mice, whereas total collagen content was 30% lower (p < 0.01) and the cumulative score of aortic lesions was eight times higher than in wild-type (WT) mice (p < 0.01). After 9 months of doxycycline treatment, MMP-9 activity, collagen content, and lesions in the aortas of HT mice were at the level of those of WT mice (p > 0.05). In the mouse model of collagen III haploinsufficiency treatment with broad-spectrum MMP inhibitor that was started early in life normalized increased MMP activity, reduced aortic collagen content in adults, and prevented the development of spontaneous aortic lesions. Our findings provide experimental justification for the clinical evaluation of the benefit of doxycycline at least in the haploinsufficient variety of vEDS.

Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts.

The vascular type of the Ehlers-Danlos syndrome (vEDS) is caused by dominant-negative mutations in the procollagen type III (COL3A1) gene. Patients with this autosomal dominant disorder have a shortened life expectancy due to complications from ruptured vessels or hollow organs. We tested the effectiveness of allele-specific RNA interference (RNAi) to reduce the mutated phenotype in fibroblasts. Small-interfering RNAs (siRNAs) discriminating between wild-type and mutant COL3A1 allele were identified by a luciferase reporter gene assay and in primary fibroblasts from a normal donor and a patient with vEDS. The best discriminative siRNA with the mutation at position 10 resulted in >90% silencing of the mutant allele without affecting the wild-type allele. Transmission and immunogold electron microscopy of extracted extracellular matrices from untreated fibroblasts of the patient with vEDS revealed structurally abnormal fibrils. After siRNA treatment, collagen fibrils became similar to fibrils from fibroblasts of normal and COL3A1 haploinsufficient donors. In addition, it was shown that expression of mutated COL3A1 activates the unfolded protein response and that reduction of the amount of mutated protein by siRNA reduces cellular stress. Taken together, the results provide evidence that allele-specific siRNAs are able to reduce negative effects of mutated COL3A1 proteins. Thus, the application of allele-specific RNAi may be a promising direction for future personalized therapies to reduce the severity of vEDS.

Doxycycline ameliorates the susceptibility to aortic lesions in a mouse model for the vascular type of Ehlers-Danlos syndrome.

The vascular form of Ehlers-Danlos syndrome (vEDS), a rare disease with grave complications resulting from rupture of major arteries, is caused by mutations of collagen type III [α1 chain of collagen type III (COL3A1)]. The only, recently proven, preventive strategy consists of the reduction of arterial wall stress by ß-adrenergic blockers. The heterozygous (HT) Col3a1 knockout mouse has reduced expression of collagen III and recapitulates features of a mild presentation of the disease. The objective of this study was to determine whether changing the balance between synthesis and degradation of collagen by chronic treatment with doxycycline, a nonspecific matrix metalloproteinase (MMP) inhibitor, could prevent the development of vascular pathology in HT mice. After 3 months of treatment with doxycycline or placebo, 9-month-old HT or wild-type (WT) mice were subjected to surgical stressing of the aorta. A 3-fold increase in stress-induced aortic lesions found in untreated HT mice 1 week after intervention (cumulative score 4.5 ± 0.87 versus 1.3 ± 0.34 in WT, p < 0.001) was fully prevented in the doxycycline-treated group (1.1 ± 0.56, p < 0.001). Untreated HT mice showed increased MMP-9 activity in the carotid artery and decreased collagen content in the aorta; however, in doxycycline-treated animals there was normalization to the levels observed in WT mice. Doxycycline treatment inhibits the activity of tissue MMP and attenuates the decrease in the collagen content in aortas of mice haploinsufficient for collagen III, as well as prevents the development of stress-induced vessel pathology. The results suggest that doxycycline merits clinical testing as a treatment for vEDS.

Dermal fibroblasts promote the migration of dendritic cells.

Migration of dendritic cells (DCs) from skin to lymph nodes on activation is an essential step in the initiation of an adequate immune response. The dermal microenvironment including stromal cells and their soluble factors might be involved in the regulation of DC migration. To focus on the role of dermal fibroblasts, we studied whether interaction of DCs with fibroblasts promotes the migration of DCs. DCs were co-cultured with resting fibroblasts or with tumor necrosis factor (TNF)alpha/IL-1beta-activated fibroblasts to mimic an inflammatory microenvironment. Interaction of DCs with TNFalpha/IL-1beta-stimulated fibroblasts increased the secretion of matrix metalloproteinase-9 (MMP-9) from DCs within 6 hours compared with DCs alone or DCs stimulated with lipopolysaccharide or TNFalpha/IL-1beta. In contrast, unstimulated fibroblasts did not affect MMP-9 secretion. IL-6 released by TNFalpha/IL-1beta-stimulated fibroblasts was identified as a factor responsible for fibroblast-stimulated MMP-9 secretion from DCs. In accordance with the elevated MMP-9 release, on co-culture with TNFalpha/IL-1beta-stimulated fibroblasts, DCs migrated significantly more effectively through matrigel matrices than did TNFalpha/IL-1beta-stimulated DCs. This was inhibited by a selective blocking of MMP-9, indicating the importance of MMP-9 for this migratory capacity of DCs. In summary, fibroblasts in the local dermal microenvironment are capable of potentiating the migratory capacity of DCs, and thus have the potential to actively participate in the regulation of a cutaneous immune response.

Ribose treatment reduced the infarct size and improved heart function after myocardial infarction in rats.

OBJECTIVE: In this study the effect of ribose on heart function and infarct-size was analyzed 6 h after myocardial infarction (MI) in rats. METHODS: Continuous i.v.-infusion of NaCl or ribose (200 mg/kg/h) was started one day prior to induction of MI in female Sprague-Dawley rats which was done by ligation of the left coronary artery. Six hours after MI heart function was measured with 3F tip catheter, cardiac output by thermodilution method. Thereafter the ischemic area was delineated by Evans Blue infusion, and the infarct area was visualized by triphenyltetrazolium chloride staining. The mRNA expression of interleukin (IL)-1beta, IL-6, matrix-metalloproteinase (MMP)-8, and -9 was measured by ribonuclease protection assay. RESULTS: Heart function was severely depressed 6 hours after coronary artery occlusion, but recovered significantly under the influence of ribose. Left ventricular (LV) systolic pressure (LVSP) and contractility (LVdP/dtmax) were restored to the normal levels of sham-operated animals, while parameters of LV relaxation (LVdP/dtmin and time constant of relaxation tau) were impaired compared to sham-operated animals, but significantly improved by ribose treatment compared to sham-treated MI-rats. Moreover, the infarct size was significantly smaller in the ribose treated animals despite a comparable ischemic area at risk in all MI-rats. The cytokine mRNA expression after MI was significantly reduced after ribose treatment, while there were no differences regarding MMP expression. CONCLUSION: MI size was significantly reduced and LV function significantly improved by ribose treatment at 6 h after MI. This seemed to be based on slowing the velocity of the necrotic wave front across the LV wall after MI resulting in smaller infarcts.

Crucial role of interleukin-6 in the development of norepinephrine-induced left ventricular remodeling in mice.

BACKGROUND: Elevated serum concentration of interleukin (IL)-6 is a predictor for poor prognosis in congestive heart failure. It was shown previously in rats, that IL-6 expression in the left ventricle (LV) was followed by LV hypertrophy. METHODS: Using IL-6 deficient mice (IL-6(-/-)), we studied the role of IL-6 in a model of norepinephrine (NE)-induced LV hypertrophy. RESULTS: In wild type (WT) mice, IL-6 mRNA expression and its concentration in the serum were elevated after 4 h of NE-treatment (s.c. 0.25 mg.h)./kg Further, NE-induced LV hypertrophy was detected: LV weight/body weight (LVW/BW) ratio (+12.3+/-3%, p < 0.05) and mRNA expression of atrial natriuretic peptide (ANP) in WT mice (+120+/-25%, p < 0.05) after 3 days were increased. In contrast, NE did not induce elevation of LVW/BW ratio and ANP expression in IL-6(-/-) mice. Replacement with recombinant IL-6 restored the hypertrophy-inducing effect of NE in IL-6(-/-) mice. As to the extracellular matrix (ECM) proteins, NE increased collagen type I and III expression only in WT mice and not in IL-6(-/-) mice. The addition of recombinant IL-6 elevated the expression of the ECM proteins to the WT level. CONCLUSION: IL-6 is a major player in the development of NE-induced LV hypertrophy in mice.

Cord blood cell therapy alters LV remodeling and cytokine expression but does not improve heart function after myocardial infarction in rats.

OBJECTIVE: In this study the ability of unrestricted somatic stem cells (USSC) and mononuclear cord blood cells (MN-CBC) was tested to improve heart function and left ventricular (LV) remodeling after myocardial infarction (MI). METHODS: The cells were delivered by i.v. or intramyocardial injections in rat models of MI by permanent coronary artery occlusion and by ischemia/reperfusion (I/R) injury. Heart function and remodeling was followed by recurrent echocardiography over 8 or 12 weeks after which catheterization was performed. RESULTS: Although injected labeled cells could be observed within the myocardium for up to 6 d, there was no sign of cardiac regeneration 8 or 12 weeks after MI. However, the mRNA expression of components of the extracellular matrix was attenuated in the infarct scar 12 weeks after MI and cell injection. Additionally, the expression of interleukin (IL)-6 but not of IL-1 beta increased at the site of injury and the adjacent border-zone 12 weeks after I/R and USSC-injection. However, these effects did not translate into improved heart function or attenuated LV dilatation. CONCLUSION: These data indicate that cord blood cell implantation after MI acts through paracrine mechanisms to modify remodeling rather than myocyte regeneration. The role of myofibroblasts and the optimal conditions of cell application need to be determined to translate these mechanisms into functional improvement.

Tissue inhibitor of matrix metalloproteinase-1 in norepinephrine-induced remodeling of the mouse heart.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in myocardial remodeling. Their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The present study analyzed the contribution of changes in functional and molecular parameters to early cardiac remodeling in mice hearts. The role that TIMPs might play in this process was specially acknowledged. METHODS: The remodeling was induced by norepinephrine (NE) given sc in balb/c mice. Varying concentrations, time and the addition of a neutralizing TIMP-1 antibody were evaluated. RESULTS: High dose NE led to insufficiency of the left ventricle (LV) as evidenced by reduced NE-induced elevation of LV systolic pressure, contractility and relaxation. Further, signs of lung congestion were seen. NE induced a concentration-dependent increase of LV weight/body weight (LVW/BW) ratio and elevated mRNA expression of atrial natriuretic peptide (ANP). This was accompanied by induction of collagen type I and III, as well as TIMP-1 expression. CONCLUSIONS: The NE-induced increase of TIMP-1 expression may induce the elevation of the antihypertrophic cardiac factor ANP since NE-induced increase of ANP expression was abolished by neutralizing TIMP-1 antibody. Thus, TIMP-1 may mediate ANP-induced attenuation of NE-induced hypertrophy in the mouse heart.

Cyclical mechanical stretch modulates expression of collagen I and collagen III by PKC and tyrosine kinase in cardiac fibroblasts.

Mechanical load and chemical factors as stimuli for the different pattern of the extracellular matrix (ECM) could be responsible for cardiac dysfunction. Since fibroblasts can both synthesize and degrade ECM, ventricular fibroblasts from adult rat hearts underwent cyclical mechanical stretch (CMS; 0.33 Hz) by three different elongations (3%, 6%, 9%) and four different serum concentrations (0%, 0.5%, 5%, 10%) within 24 h. Expression of collagen I and III, as well as matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), and colligin were analyzed by RNase protection assay. In the absence of serum, 9% CMS increased the mRNA of collagen I by 1.70-fold and collagen III by 1.64-fold. This increase was prevented by the inhibition either of PKC or of tyrosine kinase but not of PKA. Inhibition of PKC or tyrosine kinase itself reduced the expression of collagen I and collagen III mRNA. The mRNA of MMP-2, TIMP-2, and colligin showed the same tendency by stretch. Combined with 10% serum, 6% CMS reduced the mRNA of collagen I (0.62-fold) and collagen III (0.79-fold). Inhibition of PKC or tyrosine kinase, but not of PKA, prevented the reduction of collagen I and collagen III mRNA in 10% serum. The results show that the response of fibroblasts to CMS depends on the serum concentration. At least two signaling pathways are involved in the stretch-induced ECM regulation. Myocardial fibrosis due to ECM remodeling contributes to the dysfunction of the failing heart, which might be attributed to changes in hemodynamic loading.

Heart function and molecular biological parameters are comparable in young adult and aged rats after chronic myocardial infarction.

OBJECTIVE: To test the hypothesis that IL-1beta and IL-6 play a pivotal role after myocardial infarction (MI) particularly in aged rats. METHODS: Chronic MI was induced in young adult (3.5 months) and aged (18 months) female Sprague-Dawley rats by ligation of the left coronary artery. Sham-operated animals of corresponding age served as controls. Heart function was measured by catheterization 4 weeks after MI. The expression of IL-1beta, IL-6, TGF-beta-isoforms, ANF, and components of the extracellular matrix (pro-collagen I and III, colligin, MMP-2 and TIMP2) was measured by ribonuclease protection assay. RESULTS: Aged control rats differed from young adult rats in that LV-developed pressure (LVDP) was higher (161 vs. 147 mmHg, p<0.05) in response to the elevated total peripheral resistance (0.71 vs. 0.47 mmHg ml min/kg, p<0.05). Contractility was reduced in aged controls as indicated by decreased LV dP/dt (8.106 vs. 10.606 mmHg/s, p<0.05). LV function was severely depressed in both MI groups (reduction in LVDP by about 35% and LV dP/dt by about 30%, increase in LVEDP to 24 mmHg) while RVP and RV dP/dt markedly increased by about 100%. This was not different between both MI groups. ANF expression as a marker of hypertrophy was induced in both MI groups, but less pronounced in the LV of aged rats. Also, the mRNA expression pattern was qualitatively comparable, but showed gradual differences. CONCLUSION: These results indicate that aged rats compensate well for hemodynamic overload induced by MI. Also, the mechanisms of myocardial post-MI remodeling are comparable in young adult and aged rats.

Hematopoietic stem cells do not repair the infarcted mouse heart.

OBJECTIVE: Recent reports suggest that hematopoietic stem cells (HSC) can transdifferentiate into cardiomyoctes and contribute to myocardial regeneration after injury. This concept has recently been challenged by studies in which bone-marrow (BM)-derived cells do not acquire a cardiac phenotype after direct injection into ischemic myocardium. METHODS: In this study, we analyzed the effect of increased circulating adult BM cells by stimulation with stem cell factor (SCF; 200 microg/kg/d for 7 days) and granulocyte-colony stimulating factor (G-CSF, 50 microg/kg/d for 7 days) or by peripheral delivery of isolated adult BM cells on morphological and hemodynamic parameters of mouse hearts 6 weeks after induction of chronic myocardial infarction (MI). All animals were splenectomized to prevent sequestration of BM cells 2 weeks prior to the induction of MI. Cytokine treatment was initiated either 3 days prior to or 6 h after MI. Isolated, either whole or by magnetic beads lineage-depleted BM cells were injected via a tail vein 6 h after MI. RESULTS: Left and right ventricular (LV and RV) function revealed no improvement in any treatment group when compared to untreated MI animals at baseline resting conditions as well as after stimulation with norepinephrine (NE; 1, 5, 10, 25, 50, and 100 ng bolus i.v. in 10 microl each) as measured by catherization with ultraminiature 1.4 F tip pressure transducers 6 weeks after MI. Moreover, there was no sign of myocardial regeneration in histological or gene expression analyses. CONCLUSION: Mobilization or i.v. injection of BM cells do not have a measurable effect on cardiac regeneration.

Norepinephrine-induced changes in cardiac transforming growth factor-beta isoform expression pattern of female and male rats.

Transforming growth factor-beta (TGF-beta) is a ubiquitous growth-regulating protein with an essential role in tissue repair and formation of extracellular matrix (ECM). To better understand the role of different isoforms of TGF-beta in the cardiac remodeling process induced by norepinephrine (NE), the expression of TGF-beta1, TGF-beta2, and TGF-beta3 was studied and compared with the expression of collagen. NE (0.1 mg/kg. h) was intravenously infused in female and male Sprague-Dawley rats for several time periods, and freshly obtained ventricular myocardium after 1 day was dissociated into myocyte and nonmyocyte fractions. Prazosin (0.1 mg/kg x h) and metoprolol (1 mg/kg. h) were used to block alpha- and beta-adrenoceptors, respectively. After NE infusion, the three isoforms of TGF-beta were differentially induced as far as the magnitude and the time course is concerned. The increased expression of TGF-beta2 started earlier with a maximum after 12 hours and was more pronounced (10-fold elevation) than that of the other two isoforms, with a clear specificity for the left ventricle in female hearts. This specificity was also seen in male rats with 16-fold elevation of TGF-beta2 after 1 day of NE-stimulation. The increase of TGF-beta2 was significant only in the myocyte fraction obtained from female as well as from male hearts. The expression of the mRNA of all TGF-beta isoforms of collagen type I and type III, and of the matrix metalloproteinase (MMP)-2 and its inhibitor TIMP-2 was reduced predominantly by alpha-adrenoceptor blockade with prazosin. The increase in TGF-beta isoforms correlated with that of the mRNA expression of collagens, MMP-2 and TIMP-2.

Cardiac remodeling in erythropoietin-transgenic mice.

BACKGROUND: Transgenic (tg) mice with chronic overexpression of the human erythropoietin gene are characterized by an increased hematocrit of about 0.80 in adulthood. This is accompanied by cardiac dysfunction and premature death. The aim of this study was to examine whether this cardiac dysfunction was accompanied by hypertrophy of the heart with remodeling of the extracellular matrix (ECM). METHODS: 3-months-old wild type (wt) and tg mice without cardiac hypertrophy were compared with the respective 7-months-old mice. The mRNA of brain natriuretic peptide (BNP), of the matrix metalloproteinases (MMP)-2, -8, -9, -13, of the tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, -4 and of collagen I and III was detected by ribonuclease protection assay. The activity of MMPs was measured by zymography. RESULTS: There was hypertrophy of both ventricles in 7-months-old tg mice, which was accompanied by elevated mRNA expression of BNP. MMP-2 activity was increased and MMP-9 activity was decreased in the left ventricle (LV) of 3-months-old tg mice. This was accompanied by elevated TIMP-4 expression, followed by a shift of collagen mRNA expression from type III to type I in this ventricle. CONCLUSION: The shift to collagen I in the heart of tg mice might be associated with a stiffer ventricle resulting in diastolic dysfunction. This may be responsible for a relative and intermittent LV- and right ventricle (RV)-insufficiency which was likely to have occurred as evidenced by the elevation of lung and liver weight with hemorrhage and interstitial fibrosis after 7 months.

Norepinephrine-induced acute heart failure in transgenic mice overexpressing erythropoietin.

OBJECTIVE: Overexpression of erythropoietin (Epo) in mice (Epo-tg6) leads to an increase in hematocrit and blood volume, and strongly reduces endurance upon exercise. It was the aim of this study to characterize the mechanisms underlying the reduced cardiac performance. METHODS: Left (LV) and right (RV) ventricular function was measured with and without norepinephrine (NE) stimulation in 12 anaesthetized Epo-tg6 and in 13 wild-type (WT) control mice. RESULTS: There were no differences in heart function under baseline resting conditions. Stimulation with NE (10 microl bolus injections of 1-100 ng per mouse) in WT mice led to a dose-dependent increase in heart rate (HR), LV developed pressure (LVDP) and rate of rise in LV pressure (LV dP/dt(max)), while LV end-diastolic pressure (LVEDP) was unchanged. Except for HR, these parameters increased to a lesser extent in EPO-tg6 mice. Strikingly, LVEDP strongly increased in Epo-tg6 mice after NE (up to >20 mmHg). Eleven out of 13 Epo-tg6, but none of the WT mice died or required resuscitation after high-doses of NE. In these cases severe diastolic dysfunction became overt since the relative myocardial relaxation time was significantly prolonged and the duration of diastole was shortened. Moreover, the ECG showed a marked ST segment depression as well as deep negative T-waves. The NE-induced reduction in myocardial adenosin-triphosphate (ATP) content was more pronounced in Epo-tg6 mice after 10 min of continuous NE infusion (50 ng/min per mouse). CONCLUSION: NE-induced stress in Epo-tg6 mice led to acute heart failure associated with diastolic dysfunction and myocardial ischemia.

Norepinephrine-induced cardiac hypertrophy and fibrosis are not due to mast cell degranulation.

The norepinephrine (NE)-induced hypertrophy of the left ventricle (LV) in the rat is preceded by increased interleukin (IL)-6 expression and associated with LV fibrosis. We have examined whether the elevated level of IL-6 may be due to mast cell degranulation. Therefore we tested the effect of cromoglycate sodium salt (cromolyn), an inhibitor of mast cell degranulation with anti-inflammatory and membrane-stabilizing activity, on the increased expression of IL-6 mRNA and of mRNAs of proteins involved in the remodelling of the extracellular matrix (ECM) which is induced by NE (0.1 mg/kg x h). After 4 h, the NE-induced increase in IL-6 mRNA expression was not influenced by cromolyn (20 mg/kg x h). Cromolyn-infusion for 3 days did not affect the extent of LV hypertrophy induced by NE, as measured by the LV weight/body weight (LVW/BW) ratio and by atrial natriuretic peptide (ANP) expression. Cromolyn induced a slight depression of the NE-induced elevation of the matrix metalloproteinase (MMP)-2. However, it did not affect the NE-induced elevated levels of mRNAs of collagen I and III and the tissue inhibitor of matrix metalloproteinase (TIMP)-2. Since cromolyn did not reduce the NE-effects in rat hearts in vivo we conclude that mast cell degranulation seems not to be involved in them.

Effect of propranolol on cardiac cytokine expression after myocardial infarction in rats.

The pro-inflammatory cytokines interleukin (IL)-1beta and IL-6 have been shown to be upregulated in the myocardium after injury and after adrenergic receptor stimulation. Together with other cytokines, such as the transforming growth factor (TGF)-beta, the pro-inflammatory cytokines have been implicated in the initiation of tissue repair and wound healing after myocardial infarction (MI). In the present study, the effect of beta-adrenergic receptor blockade with propranolol (2 mg/kg x h s.c. by miniosmotic pumps) on cardiac cytokine expression and on wound healing was analyzed in rats from 6-72 h after MI. IL-1beta and IL-6 gene expression strongly increased in the infarcted myocardium 6 h after MI and peaked after 12 h, while TGF-beta, progressively increased from 12 h onwards. Also, TGF-beta2 increased after 12 h, peaked after 24 h and declined thereafter, while TGF-beta, was only elevated after 72 h. Treatment with propranolol had a negative chronotropic effect throughout the observation period of 72 h. It attenuated the initial elevation in LVEDP and increased cardiac output ultimately. Furthermore, propranolol attenuated IL-1beta mRNA expression, but had not effect on the other cytokines. Moreover, MMP-9 gelatinolytic activity was markedly attenuated by propranolol indicating a delayed resorption of the necrotic tissue and, possibly, collagen turnover. Replacement by scar tissue, however, was not affected as indicated by normal collagen expression.

Pulmonary edema and pleural effusion in norepinephrine-stimulated rats--hemodynamic or inflammatory effect?

Stimulation with norepinephrine (NE) leads to pulmonary edema and pleural effusion in rats. These pulmonary fluid shifts may result from pulmonary congestion due to the hemodynamic effects of NE and/or inflammation with an increase in vascular permeability. The contribution of these two factors were investigated in the present study. Female Sprague-Dawley rats received continuous i.v. NE infusion (0.1 mg/kg/h) over time intervals between 90 min and 72 h. After heart catheterization, pleural fluid (PF) and lung tissue were obtained. In some of the animals, a bronchoalveolar lavage (BAL) was performed. Pulmonary edema and inflammation were shown histologically. We determined the expression of interleukin (IL)-6 as one of the most potent acute-phase protein mediators in serum, PF and BAL supernatant fluid (BALF) using ELISA as well as in the lung tissue using Western blotting. Total protein concentration in BALF and PF served as indicators of increased capillary permeability. Pulmonary edema and pleural effusion appeared coincidentally with an increase in total peripheral resistance (TPR) after 6 h of NE infusion. PF reached a maximum between 8 and 16 h (2.2 +/- 0.3 ml, controls < 0.5 ml) and disappeared within 48 h. Activation of IL-6 in the fluids was observed after 8 h of NE stimulation. In the lung tissue it started after 12 h and reached 330% of the control value after 48 h. Pulmonary inflammation was documented histologically. It was accompanied by increased protein concentration in BALF after 24 h of NE treatment. Hemodynamic effects of NE are the main causative factors in the initial phase of the pulmonary fluid shifts. Additionally, NE leads to an activation of cytokines such as IL-6 and to inflammation and to an increase in capillary permeability. However, inflammation and increased capillary permeability occurred later than pulmonary edema and pleural effusion. Hence, we conclude that they are secondary factors which may contribute to maintain the fluid shifts over a longer period of time.

The expression of mRNA of cytokines and of extracellular matrix proteins in triiodothyronine-treated rat hearts.

In various models of cardiac hypertrophy, e.g. treatment of rats with norepinephrine infusion or pressure overload, increased expression of cytokines together with increase in extracellular matrix proteins (ECMP) was reported. In this study the effect of triiodothyronine (T3) on the expression of mRNA for cytokines and ECMP was investigated. Female Sprague-Dawley rats were treated daily with T3 in a dose of 0.2 mg x kg(-1) of body weight s.c. Changes in the left (LV) and right (RV) ventricular function were measured 6, 24, 48, 72 h and 7 and 14 days after the first T3-injection using Millar ultraminiature pressure catheter transducers. RNA was isolated from LV and RV tissue, and the expression of cytokines and ECMP was measured using the ribonuclease protection assay. T3-treatment induced a significant increase in LV dP/dtmax and RV dP/dtmax, (p < 0.05) 24 h after the first injection of T3 together with an increase in heart rate (p < 0.01). The RV systolic pressure increased 48 h after the first T3 injection, whereas the LV systolic pressure remained unchanged. After 48 h the heart weight to body weight ratio was increased (p < 0.01). Hypertrophy of the RV was more prominent than that of the LV (155.9 vs. 137.7%). In all groups the expression of mRNA for interleukins (IL) IL-6, IL-1beta, IL-1alpha and tumour necrosis factor (TNF)-alpha in both ventricles did not change (p > 0.05). There was a significant increase in the mRNA for colligin 24 h after the T3 injection in both LV (p < 0.01) and RV (p < 0.05). This was followed by an increase in the mRNA for collagen I and III 72 h after the first T3-dose (p < 0.05 in RV; p < 0.01 in LV). At this point, the mRNA for tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was increased (p < 0.01) in the LV only. Moreover, after 7 days also the mRNA for matrix metalloproteinase (MMP)-2 increased (p < 0.01) in the LV. Both, TIMP-2 and MMP-2 were increased in the RV only after 14 days (p < 0.05). The gelatinase activity of MMP-2, however, was unchanged in both ventricles. The T3-induced cardiac hypertrophy was not accompanied by fibrosis as measured by the Sirius red staining after 14-days of T3-treatment. The moderate increase in mRNA for ECMP and MMP may be attributed more to the increasing mass of the ventricles with the accompanying remodelling of the ECM than to increased fibrosis.

Norepinephrine-induced expression of cytokines in isolated biventricular working rat hearts.

The norepinephrine (NE)-induced hypertrophy of the left ventricle (LV) in the rat is associated with increased interleukin (IL)-6 and IL-1beta expression. In the present study, a newly established model of isolated biventricular working rat heart was used to examine whether NE may directly induce cytokine mRNA expression in a preparation devoid of other circulating hormonal and humoral factors. Representative hemodynamic parameters and the expression of various cytokines of the isolated biventricular working heart (IBWH) were compared with the respective in vivo results. Systolic pressure (SP) of the right ventricle (RVSP) was higher in the IBWH than in the intact anesthetized rat (42.9 +/- 1.89 vs. 32.3 +/- 1.06). However, heart rate (HR), LVSP and the maximal rate of pressure development of LV (LV dP/dt(max)) were lower. After NE infusion (30 nM), SP and dP/dt(max) were increased by 30 and 90%, respectively, in both ventricles. In vivo, the ventricles showed a different response to NE (0.1 mg/kg x h): LVSP increased by 15%, RVSP and RV dP/dt(max) was doubled, LV dP/dt(max) was tripled. The analysis of cytokine mRNA expression with the RNase protection assay revealed that in vivo IL-6 and IL-1beta were increased between 4 and 12 h 80- and 12-fold, respectively, while there was weak expression under control conditions. In the IBWH IL- 1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF)alpha were increased already during control perfusion. The increase of these stress-activated cytokines indicates that the isolation and perfusion procedure may exert a stress on the heart. NE induced an additional time-dependent increase of IL-6 mRNA after 1 h of infusion. Thus, NE has a direct effect on the cardiac IL-6 expression, which occurred earlier in the in vitro preparation than in the rat heart in vivo.

Catecholamine-induced pulmonary edema and pleural effusion in rats--alpha- and beta-adrenergic effects.

We investigated the contribution of alpha- and beta-adrenergic pathways to catecholamine-induced pulmonary edema and the role of pleural effusion in preventing alveolar edema. Female Sprague-Dawley rats received continuous intravenous infusion of norepinephrine and of separate alpha- or beta-adrenergic stimulation over 6-24 h. We performed heart catheterization in vivo and excised post mortem lung tissue for histological analysis. Interleukin (IL)-6 and total protein concentrations were determined in serum, pleural fluid (PF) and bronchoalveolar lavage fluid. alpha-Adrenergic treatment increased right ventricular systolic pressure (RVSP) and total peripheral resistance (TPR) and caused severe alveolar edema associated with IL-6 activation in serum and diffuse pulmonary inflammation. PF amounts were moderate (0.9+/-0.2 ml). beta-Adrenergic stimulation also increased RVSP but decreased TPR. Interstitial but not alveolar edema and focal inflammation without IL-6 activation developed. Large PF amounts (6.2+/-1.5 ml) occurred which were considered to prevent alveolar edema. We conclude that both alpha- and beta-adrenergic stimulation contribute to pulmonary fluid shifts in rats, but alpha-adrenergic pathways cause more acute and more severe lung injury than beta-adrenergic mechanisms.