Lungs Of Ferrets Research Articles

How should airway smooth muscle be punished for causing asthma?

asthma is a complex disease that has varied manifestations, is often difficult to treat, and indeed is even a challenge to define. Nevertheless, there are some features of asthma that are characteristic. Key among these is airways hyperresponsiveness (AHR), defined as an abnormally large mechanical

Chronic inflation of ferret lungs with CPAP reduces airway smooth muscle contractility in vivo and in vitro

The mechanical stress imposed on the lungs during breathing is an important modulator of airway responsiveness in vivo. Our recent study demonstrated that continuous positive airway pressure applied to the lungs of nonanesthetized, tracheotomized rabbits for 4 days decreased lower respiratory system responsiveness to challenge with ACh (Xue Z, Zhang L, Ramchandani R, Liu Y, Antony VB, Gunst SJ, Tepper RS. J. Appl Physiol 99: 677-682, 2005). In addition, airway segments excised from the lungs of these animals and studied in vitro exhibited reduced contractility. However, the mechanism for this reduction in contractility was not determined. The stress-induced decrease in airway responsiveness could have resulted from alterations in the excitation-contraction coupling mechanisms of the smooth muscle cells, or it might reflect changes in the structure and/or composition of the airway wall tissues. In the present study, we assessed the effect of prolonged chronic stress of the lungs in vivo on airway smooth muscle force generation, myosin light chain phosphorylation, and airway wall structure. To enhance the potential development of stress-induced structural changes, we applied mechanical stress for a prolonged period of time of 2-3 wk. Our results demonstrate a direct connection between the decreased airway responsiveness caused by chronic mechanical stress of the lungs in vivo and a persistent decrease in contractile protein activation in the airway smooth muscle isolated from those lungs. The chronic stress also caused an increase in airway size but no detectable changes in the composition of the airway wall.

Toxicological evaluation of live attenuated, cold-adapted H5N1 vaccines in ferrets

Live attenuated influenza vaccines (LAIV) have several attributes related to safety, immunogenicity, cross-protection against antigenic drift strains, high yield and needle-free administration that make them attractive candidates for control of pandemic influenza. H5N1 LAIV vaccine candidates are attenuated in ferrets, chickens and mice. These vaccine candidates were further characterized in the ferret model to evaluate their toxicity at doses comparable to seasonal LAIV and at doses up to 100-fold higher. The results demonstrated that H5N1 LAIV, even when administered at high doses, is restricted in replication in the lower respiratory tract of ferrets. However, intranasal administration of 0.5 mL can result in deposition of H5N1 LAIV in the ferret lung, where it induces a pulmonary inflammatory response in the absence of significant local replication of the vaccine virus. Thus, smaller vaccine dose volumes should be considered for evaluation of LAIV in animal models.

Severe Acute Respiratory Syndrome Coronavirus Infection in Vaccinated Ferrets

Background. Development of vaccines to prevent severe acute respiratory syndrome (SARS) is limited by the lack of well-characterized animal models. Previous vaccine reports have noted robust neutralizing antibody and inflammatory responses in ferrets, resulting in enhanced hepatitis. Methods. We evaluated the humoral immune response and pathological end points in ferrets challenged with the Urbani strain of SARS-associated coronavirus (SARS-CoV) after having received formalin-inactivated whole-virus vaccine or mock vaccine. Results. Humoral responses were observed in ferrets that received an inactivated virus vaccine. Histopathological findings in lungs showed that infection of ferrets produced residual lung lesions not seen in both mock and vaccinated ferrets. SARS-CoV infection demonstrated bronchial and bronchiolar hyperplasia and perivascular cuffing in ferret lung tissue, as seen previously in infected mice. No evidence of enhanced disease was observed in any of the ferrets. All of the ferrets cleared the virus by day 14, 1 week earlier if vaccinated. Conclusions. The vaccine provided mild immune protection to the ferrets after challenge; however, there was no evidence of enhanced liver or lung disease induced by the inactivated whole-virus vaccine. The ferret may provide another useful model for evaluating SARS vaccine safety and efficacy.

Effects of 6-month daily supplementation with oral beta-carotene in combination or not with benzo[a]pyrene on cell-cycle markers in the lung of ferrets

Epidemiological studies have demonstrated that people who eat more fruits and vegetables (rich in carotenoids) and people who have higher serum beta-carotene (BC) levels have a lower risk of cancer, particularly lung cancer. However, the two main human intervention studies of BC supplementation (the ATBC and the CARET trials) revealed an increased risk of lung cancer among smokers and asbestos workers. Previous studies carried out in the ferret have reported that BC effects are related to dose. Here, we treated ferrets with two concentrations of oral BC (0.8 and 3.2 mg/kg body weight per day) for 6 months, using BC in a formulation also containing dl-alpha-tocopherol and ascorbyl palmitate. The effect of the smoke-derived carcinogenic agent benzo[a]pyrene (BP), with or without low-dose BC, was also analysed. We determined the protein levels and mRNA expression levels of activator protein 1 (c-Jun and c-Fos), c-Myc, cyclin D1, proliferating cellular nuclear antigen and retinoic acid receptor beta. We did not find higher levels of cell proliferation markers in the lung of ferrets treated with BC or signals of squamous metaplasia lesions either. On the other hand, although no evident signals of pulmonary carcinogenesis were observed in animals exposed to BP, BC supplementation in these animals may prevent against excess cell proliferation, since this reestablishes Jun protein and cyclin D1 mRNA levels in the lung of BP-exposed animals. In summary, these results show that the combination of BC with alpha-tocopherol and ascorbyl palmitate does not induce pro-oxidant effects in the lung of ferrets.

Genotoxic effects induced by combined exposure to beta-carotene and benzo[ a]pyrene in ferret lungs

Evidence from both epidemiological and experimental observations have fueled the belief that the high consumption of fruits and vegetables rich in carotenoids may help prevent cancer and heart disease in humans. Because of its well-documented antioxidant and antigenotoxic properties, the carotenoid β-carotene (βCT) gained most of the attention in the early 1980s and became one of the most extensively studied cancer chemopreventive agents in population-based trials supported by the National Cancer Institute. However, the results of three randomized lung cancer chemoprevention trials on βCT supplementation unexpectedly contradicted the large body of epidemiological evidence relating to the potential benefits of dietary carotenoids. Not only did βCT show no benefit, it was associated with significant increases in lung cancer incidence, cardiovascular diseases, and total mortality. These findings aroused widespread scientific debate that is still ongoing. It also raised the suspicion that βCT may even possess co-carcinogenic properties. In this review, we summarize the current data on the co-carcinogenic properties of βCT that is attributed to its role in the induction of carcinogen metabolizing enzymes and the over-generation of oxidative stress. The data presented provide convincing evidence of the harmful properties of this compound if given alone to smokers, or to individuals exposed to environmental carcinogens, as a micronutrient supplement. This has now been directly verified in a medium-term cancer transformation bioassay. In the context of public health policies, while the benefits of a diet rich in a variety of fruits and vegetables should continue to be emphasized, the data presented here point to the need for consideration of the possible detrimental effects of certain isolated dietary supplements, before mass cancer chemoprevention clinical trials are conducted on human subjects. This is especially important for genetically predisposed individuals who are environmentally or occupationally exposed to mutagens and carcinogens, such as those found in tobacco smoke and in industrial settings.

Systemic neovascularization of the lung after pulmonary artery occlusion: “decoding the Da Vinci code”

Five hundred years ago, Leonardo da Vinci demonstrated that new bronchial vessels can develop in a human lung. The development of a mouse model of pulmonary artery obstruction in recent years ([1][1]) has provided substantial insight into the mechanisms and consequences of the systemic

Combined antioxidant (β-carotene, α-tocopherol and ascorbic acid) supplementation increases the levels of lung retinoic acid and inhibits the activation of mitogen-activated protein kinase in the ferret lung cancer model

Interactions among beta-carotene (BC), alpha-tocopherol (AT) and ascorbic acid (AA) led to the hypothesis that using a combination of these antioxidants could be more beneficial than using a single antioxidant alone, particularly against smoke-related lung cancer. In this investigation, we have conducted an animal study to determine whether combined BC, AT and AA supplementation (AOX) protects against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis in smoke-exposed (SM) ferrets. Ferrets were treated for 6 months in the following four groups: (i) control, (ii) SM + NNK, (iii) AOX and (iv) SM + NNK + AOX. Results showed that the combined AOX supplementation (i) prevented the SM + NNK-decreased lung concentrations of retinoic acid (RA) and BC; (ii) inhibited the SM + NNK-induced phosphorylation of Jun N-terminal kinase (JNK), extracellular-signal-regulated protein kinase (ERK) and proliferating cellular nuclear antigen proteins in the lungs of ferrets; and (iii) blocked the SM + NNK-induced up-regulation of total p53 and Bax proteins, as well as phosphorylated p53 in the lungs of ferrets. In addition, there were no lesions observed in the lung tissue of ferrets in the control and/or the AOX groups after 6 months of intervention, but combined AOX supplementation resulted in a trend toward lower incidence of both preneoplastic lung lesions and lung tumor formation in SM + NNK + AOX group of ferrets, as compared with the SM + NNK group alone. These data indicate that combined AOX supplementation could be a useful chemopreventive strategy against lung carcinogenesis through maintaining normal tissue levels of RA and inhibiting the activation of mitogen-activated protein kinase pathways, cell proliferation and phosphorylation of p53.

Induction of pulmonary neoplasia in the smoke-exposed ferret by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): A model for human lung cancer

Research into dietary chemoprevention against lung carcinogenesis has been limited by the lack of appropriate animal models that closely mimic smoking-related human lung cancer. Ferrets ( Mustela putorius furo) have been used to study the biologic activities of carotenoids against smoke-induced lung lesions, but this model has yet to be thoroughly established and validated. To determine the appropriateness of the ferret as a model for human lung cancer, we have performed a 6-month in vivo study in ferrets exposed to both tobacco smoke and a carcinogen (4-( N-methyl- N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK) found in cigarette smoke. Results showed that six out 12 ferrets exposed to both NNK injection and cigarette smoke developed grossly identifiable lung tumors whereas none of nine ferrets from the sham treatment group developed any lung lesions. The histopathological types of these tumors (squamous cell carcinoma, adenosquamous carcinoma and adenocarcinoma) in ferret lungs are very similar to those in humans. In addition, 10 out of 12 ferrets exposed to both NNK and cigarette smoke developed preneoplastic lesions (squamous metaplasia, dysplasia, and atypical adenomatous hyperplasia) with complex growth patterns whereas the sham group did not show any of these lesions. Furthermore, the expression of proliferating cellular nuclear antigen increased markedly in both gross tumors and preneoplastic lesions in the lungs. In summary, the development of both preneoplastic lesions and gross lung tumors in ferrets provides an excellent and unique model for studying lung cancer chemoprevention with agents such as carotenoids, and for studying the molecular mechanism of carcinogenesis in the earlier stages of smoke-related lung cancer.

Adeno-Associated Virus Vectors in Clinical Trials

Adeno-Associated Virus Vectors in Clinical Trials

Low Dose β-Carotene Supplementation of Ferrets Attenuates Smoke-Induced Lung Phosphorylation of JNK, p38 MAPK, and p53 Proteins

We demonstrated previously that smoke exposure and/or high-dose beta-carotene supplementation decreases levels of retinoic acid and retinoic acid receptor beta (RARbeta) protein, but increase levels of c-Jun and proliferating cellular nuclear antigen protein in the lungs of ferrets. In contrast, low-dose beta-carotene can prevent the decreased lung retinoic acid and the smoke-induced lung lesions. In the present study, we investigated whether smoke exposure and/or beta-carotene supplementation could affect Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and p53 in the lungs of ferrets. Ferrets were subjected to cigarette smoke exposure and either a high or low dose of beta-carotene (2 x 3 factorial design) for 6 mo. There were greater protein levels of phosphorylated JNK, p38, and c-Jun, but lower levels of MAPK phophatase-1 (MKP-1) in groups exposed to smoke and/or high dose beta-carotene. Both phosphorylated-p53 and total p53 were substantially increased in the lungs of these groups. In contrast, low-dose beta-carotene greatly attenuated the smoke-induced phosphorylation of JNK, p38, c-Jun, p53, and total p53, accompanied by upregulated MKP-1. Smoke exposure increased MAPK kinase-4 (MKK4) phosphorylation regardless of beta-carotene supplementation. These data indicate that restoration of retinoic acid and MKP-1 by low-dose beta-carotene in the lungs of ferrets may prevent the smoke-induced activation of the JNK-dependent signaling pathway, p38 MAPK, and the associated phosphorylation of p53, thereby lowering the risk of the smoke-related lung lesions. These data provide supportive evidence that the beneficial vs. detrimental effects of beta-carotene supplementation are related to the dosage of beta-carotene administered.

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3·3 logs (95% Cl 2·6–4·0 logs; p<0·001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0·013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.

Exposing Ferrets to Cigarette Smoke and a Pharmacological Dose of β-Carotene Supplementation Enhance In Vitro Retinoic Acid Catabolism in Lungs via Induction of Cytochrome P450 Enzymes

In our previous studies, we found lower levels of retinoic acid (RA) in the lungs of ferrets exposed to cigarette smoke and/or a pharmacological dose of beta-carotene. To determine whether this is involved in excessive catabolism of RA via cytochrome P450 (CYP) induction, we carried out in vitro incubations of RA with the lung microsomal fractions of ferrets with or without CYP inhibitors and antibodies against CYP. The polar metabolites (4-oxo-RA and 18-hydroxy-RA) of RA metabolism after the incubation were analyzed by HPLC. Expressions of CYP (1A1, 1A2, 2E1 and 3A1) were examined using Western blot analysis. Incubation of various concentrations of RA with the lung microsomal fraction from ferrets exposed to cigarette smoke, a pharmacological dose of beta-carotene or their combination dose-dependently increased the levels of 4-oxo-RA and 18-hydroxy-RA compared with that of the control ferrets. At all RA concentrations, this increase was the greatest in lung tissue from the combined treatment group. Furthermore, this enhanced RA catabolism was substantially (approximately 80%) inhibited by nonspecific CYP inhibitors (disulfiram and liarozole), but was partially (approximately 50%) inhibited by resveratrol (CYP1A1 inhibitor), alpha-naphthoflavone (CYP1A2 inhibitor) and antibodies against CYP1A1 and CYP1A2. Cigarette smoke exposure and/or pharmacological doses of beta-carotene increased levels of CYP1A1 and 1A2 by three- to sixfold but not levels of 2E1 and 3A1 in ferret lung tissue. These findings suggest that low levels of RA in the lung of ferrets exposed to cigarette smoke and/or pharmacological doses of beta-carotene may be caused by the enhanced RA catabolism via induction of CYP, CYP1A1 and CYP1A2 in particular, which provides a possible explanation for enhanced lung carcinogenesis seen with pharmacological doses of beta-carotene supplementation in cigarette smokers.

Conditions for in vitro maturation and artificial activation of ferret oocytes.

The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time. In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG [10 IU/ml] and hCG [5 IU/ml]), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol [2 microg/ml]). After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%-36%, n = 67-73). At 48 h, similar maturation rates (56%-69%, n = 76-87) were observed for all three types of media. For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination. Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage. Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59). Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean +/- SEM, 10.3 +/- 1.1; n = 11) and trophectoderm (60.8 +/- 2.9, n = 11) cells. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.

The effect of low and high doses of beta-carotene and exposure to cigarette smoke on the lungs of ferrets.

When the diets of ferrets were supplemented with large (pharmacologic) daily doses of beta-carotene (BC) for 6 months, the levels of retinoic acid and the retinoic acid receptor beta declined significantly in lung tissues. Indicators of cell proliferation (c-jun and c-fos proteins and others) increased. Histologic observations showed that feeding high doses of BC resulted in keratinized squamous metaplasia in the lung tissues. When high-doses of BC were combined with daily exposure to cigarette smoke, the BC effects were greatly accentuated. These results may lead to an explanation of the increased incidence of lung cancer in two large independent epidemiologic studies of smokers in which pharmacologic doses of BC were given.

Beta-carotene and lung cancer

Abstract Does beta-carotene increase, rather than decrease, human lung cancer rates? A large body of observational epidemiologic study has demonstrated that individuals who eat more fruits and vegetables rich in carotenoids and/or who have higher levels of serum beta-carotene have a lower risk of cancer, particularly lung cancer. This inverse relationship has been particularly strong in lung cancer patients with a history of heavy smoking. However, there is contradictory evidence from recent human intervention studies using beta-carotene supplements (20­30 mg per day). An increase in risk of lung cancer among smokers who took beta-carotene supplements was reported in the Alpha Tocopherol, Beta-carotene Cancer Prevention (ATBC) Trial and among smokers and asbestos-exposed workers in the Beta-Carotene and Retinol Efficiency Trial (CARET), but not among male physicians in the United States in the Physicians Health Study (only 11 % of whom were current smokers). Whether there is a true hazard associated with beta-carotene has been evaluated in control studies using the ferret. This animal mimics the human tissue metabolism of beta-carotene, and has been used for studies of tobacco smoking and inhalation toxicology. In the first study, ferrets were given a high-dose beta-carotene supplement equivalent to 30 mg per day in humans, and exposed cigarette smoke or both for six months. A strong proliferative response in lung tissue and squamous metaplasia were observed in all beta-carotene-supplemented animals, and this response was enhanced by exposure to tobacco smoke. When compared to the control group, beta-carotene-supplemented animals (with or without smoke exposure) had statistically significantly lower concentrations of retinoic acid in lung tissue, and they exhibited reductions in RAR-beta gene expression (a tumor suppressor gene). Further, ferrets given a high-dose beta-carotene supplement and exposed to tobacco smoke had fourfold elevated expressions of c-jun and c-fos genes. In a second study, ferrets were given either physiological- or pharmacologic-dose beta-carotene supplementations, which were equivalent to 6 mg vs. 30 mg per day in humans, respectively. The animals were exposed to cigarette smoke for six months. The retinoic acid concentration and RAR beta-gene expression were reduced in the lung tissues, whereas the expression of AP1, cyclin D1, and proliferative cell nuclear antigen were greater in the high-dose, beta-carotene-supplemented animals with or without smoke, as well as the smoke-exposed, low-dose, beta-carotene-supplemented animalsbut not in the low-dose, beta-carotene-supplemented animals alone, as compared with the control group. Squamous metaplasia was only observed in the lung tissues of high-dose, beta-carotene exposed groups with or without smoke (but not the low-dose beta-carotene plus smoke group, the low-dose beta-carotene-supplemented group, or the control group). These data show that in contrast with the pharmacologic dose of beta-carotene, a physiologic dose of beta-carotene in smoke-exposed ferrets has no detrimental effectand, in fact, may afford weak protection against lung damage induced by cigarette smoke. Further studies from our laboratory have revealed an instability of the beta-carotene molecule in the lungs of cigarette smoke-exposed ferrets. Oxidized beta-carotene metabolites may play a role in lung carcinogenesis: by inducing carcinogen-bioactivating enzymes, facilitating the binding of metabolites of benz[a]pyrene to DNA, enhancing retinoic acid metabolism by P450 enzyme induction with subsequent down-regulation of RAR-beta, and acting as pro-oxidants, causing damage to DNA. Ferret studies under highly controlled experimental conditions using high- and low-dose beta-carotene in the presence of alpha tocopherol and ascorbic acid (thereby stabilizing the beta-carotene molecule) showed protective effects against smoke-induced lung squamous metaplasia in ferrets.

Genes coding for polymerase proteins are essential for attenuation of the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus

Background: The A/Leningrad/134/17/57 (H2N2) (Len/17) cold-adapted (ca) master strain used as a donor of attenuation for preparing live attenuated influenza vaccine was derived from the A/Leningrad/134/57 wild-type virus (Len/wt) after 17 passages in eggs at 25 °C. In contrast to Len/wt, the Len/17 virus is sensitive to high (40 °C) temperature ( ts), is able to grow at low (25 °C) temperature ( ca), and is attenuated ( att) for humans. Len/17 is different from Len/wt by eight coding mutations in internal genes PB2 (V-478-L), PB1 (K-265-N, V-591-I), PA (L-28-P, V-341-L), M1 (I-15-V), M2 (A-86-T) and NS2 (M-100-I). However, the role of individual mutant genes in the manifestation of the ts, ca, and att phenotypes was not established. To evaluate this, single gene reassortants (SGRs) with one mutated gene in the background of the Len/wt genome were studied. Methods: Since heterogeneity (A- or T-86 in the M2 gene) was noted in Len/17, the ts and ca clone 28 of this donor (Len/17-cl28) that has A-86 in M2 was used to prepare the SGRs. Preservation of mutations in SGRs was monitored by PCR-restriction (RFLP) analysis. Virus replication in ferret lungs was observed to evaluate the att phenotype of SGRs. Results: PB2- and PB1-SGRs each manifested the ts phenotype, and PB2-SGR demonstrated the ca phenotype as well. PA-SGR was ca, but not ts. Mutations in the M1 and NS2 genes did not affect the ts or ca phenotypes of corresponding SGRs. Ferrets infected with Len/17-cl28, Len/wt and PB2-, PB1- or PA-SGRs demonstrated comparable titers in nasal turbinates on the third day, while infectious virus was found only in the lungs of ferrets infected with Len/wt and PA-SGR. Conclusion: These data suggest that mutations in the PB2 (V-478-L) and PB1 (K-265-N, V-591-I) genes play a critical role in the attenuation of the ca Len/17 vaccine donor strain.

Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation.

Endothelial cell (EC) barrier regulation is critically dependent on cytoskeletal components (microfilaments and microtubules). Because several edemagenic agents induce actomyosin-driven EC contraction tightly linked to myosin light chain (MLC) phosphorylation and microfilament reorganization, we examined the role of microtubule components in bovine EC barrier regulation. Nocodazole or vinblastine, inhibitors of microtubule polymerization, significantly decreased transendothelial electrical resistance in a dose-dependent manner, whereas pretreatment with the microtubule stabilizer paclitaxel significantly attenuated this effect. Decreases in transendothelial electrical resistance induced by microtubule disruption correlated with increases in lung permeability in isolated ferret lung preparations as well as with increases in EC stress fiber content and MLC phosphorylation. The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase. Together, these results suggest that microtubule disruption initiates specific signaling pathways that cross talk with microfilament networks, resulting in Rho-mediated EC contractility and barrier dysfunction.

Effect of changing vascular volume on measurement of protein reflection coefficient in ischemic lungs.

In ischemic organs, the protein reflection coefficient (sigma) can be estimated by measuring blood hematocrit (Hct) and protein after increasing static vascular pressure (P(v)). Our original equation for sigma (J Appl Physiol 73: 2616-2622, 1992) assumed a constant vascular volume during convective fluid flux (). In this study, we 1) quantified the rate of vascular volume change (dV/dt) still present in ischemic single ferret lungs after 20 min of P(v) = 30 Torr and 2) developed an equation for sigma that allowed a finite dV/dt. In 25 lungs, we estimated the dV/dt after 20 min at P(v) = 30 Torr by subtracting from the rate of lung weight gain (W(L)). The relationship between (0.15 +/- 0.02 ml/min) and W(L) (0.24 +/- 0.02 g/min) was significant (R = 0.66, P < 0.001), but the slope was <1 (0.41 +/- 0.10, P < 0.05). dV/dt (0.10 +/- 0.02 ml/min) was similar in magnitude to at 20 min. The modified equation for sigma revealed that a finite dV/dt caused the original sigma measurement to underestimate true sigma. A low sigma, high, high baseline Hct, and long filtration time enhanced the error. The error was small, however, and could be minimized by adjusting experimental parameters.

Effects of physiological versus pharmacological beta-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets.

There remains a remarkable discordance between the results of observational epidemiological studies and intervention trials using beta-carotene as a potential chemopreventive agent. One question that needs to be examined is whether the adverse outcomes of human beta-carotene trials are related to the large doses of beta-carotene that were administered. In the present study, ferrets were given a physiological (low) dose or a pharmacological (high) dose of beta-carotene supplementation (0.43 mg versus 2.4 mg/kg body wt/day, which is equivalent to 6 mg versus 30 mg/day in humans) and exposed to cigarette smoke for 6 months. We investigated the effects of these doses of beta-carotene on retinoid concentrations, expression of retinoic acid receptors (RARs), activator protein 1 (AP-1; c-Jun and c-Fos), cyclin D1, proliferating cellular nuclear antigen (PCNA), and histopathological changes in the lungs of both normal and cigarette smoke-exposed ferrets. Thirty-six male ferrets were treated in six groups-control, smoke-exposed (SM), low-dose beta-carotene (LBC), high-dose beta-carotene (HBC), low-dose beta-carotene plus smoke exposure (LBC+SM) or high-dose beta-carotene plus smoke exposure (HBC+SM)-for 6 months. Retinoic acid concentration and RAR beta gene expression, but not expression of RAR alpha and RAR gamma, was reduced in the lung tissue of HBC+SM, HBC, SM and LBC+SM ferrets, but not in that of LBC ferrets, as compared with the control group. Expression of AP-1 and PCNA was greater in HBC+SM, HBC, SM and LBC+SM ferrets, but not in the LBC ferrets, as compared with the control group. Increased amounts of cyclin D1 and keratinized squamous metaplasia were observed in the lung tissue of HBC+SM, HBC and SM groups but not in that of the LBC+SM, LBC or control groups. These data suggest that, in contrast with a pharmacological dose of beta-carotene, a physiological dose of beta-carotene in smoke-exposed ferrets has no potentially detrimental effects and may afford weak protection against lung damage induced by cigarette smoke.