The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome. As a result, the role of this model is likely to become even more important. For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. An increasing number of lung conditions are being studied and modeled in the pig. Genetically modified porcine models of cystic fibrosis have been generated that, unlike mouse models, develop lung disease similar to human cystic fibrosis. However, the scientific literature relating specifically to porcine lung anatomy and airway histology is limited and is largely restricted to veterinary literature and textbooks. Furthermore, methods for in vivo lung procedures in the pig are rarely described. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model.

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Chronic hypoxia causes pulmonary hypertension with vascular remodeling, increase in vascular tone, and altered reactivity to agonists. These changes involve alterations in multiple Ca(2+) pathways in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that vanilloid (TRPV)- and melastatin-related transient receptor potential (TRPM) channels are expressed in pulmonary arteries (PAs). Here we found that TRPV4 was the only member of the TRPV and TRPM subfamilies upregulated in PAs of chronic hypoxic rats. The increase in TRPV4 expression occurred within 1 day of hypoxia exposure, indicative of an early hypoxic response. TRPV4 in PASMCs were found to be mechanosensitive. Osmo-mechanical stress imposed by hypotonic solution activated Ca(2+) transients; they were inhibited by TRPV4 specific short interfering RNA, the TRPV blocker ruthenium red, and the cytochrome P450 epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide. Consistent with TRPV4 upregulation, the Ca(2+) response induced by the TRPV4 agonist 4α-phorbol 12,13-didecanoate and hypotonicity was potentiated in hypoxic PASMCs. Moreover, a significant myogenic tone, sensitive to ruthenium red, was observed in pressurized endothelium denuded small PAs of hypoxic but not normoxic rats. The elevated basal intracellular Ca(2+) concentration in hypoxic PASMCs was also reduced by ruthenium red. In extension of these results, the development of pulmonary hypertension, right heart hypertrophy, and vascular remodeling was significantly delayed and suppressed in hypoxic trpv4(-/-) mice. These results suggest the novel concept that TRPV4 serves as a signal pathway crucial for the development of hypoxia-induced pulmonary hypertension. Its upregulation may provide a pathogenic feed-forward mechanism that promotes pulmonary hypertension via facilitated Ca(2+) influx, subsequently enhanced myogenic tone and vascular remodeling.

The systemic vasculature exhibits attenuated vasoconstriction following hypobaric chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased activity of endothelial cell (EC) large-conductance, calcium-activated potassium (BK(Ca)) channels contributes to this response. Gracilis resistance arteries from hypobaric CH (barometric pressure = 380 mmHg for 48 h) rats demonstrated reduced myogenic reactivity and hyperpolarized VSM membrane potential (E(m)) compared with controls under normoxic ex vivo conditions. These differences were eliminated by endothelial disruption. In the presence of cyclooxygenase and nitric oxide synthase inhibition, combined intraluminal administration of the intermediate and small-conductance, calcium-activated K(+) channel blockers TRAM-34 and apamin was without effect on myogenic responsiveness and VSM E(m) in both groups; however, these variables were normalized in CH arteries by intraluminal administration of the BK(Ca) inhibitor iberiotoxin (IBTX). Basal EC E(m) was hyperpolarized in arteries from CH rats compared with controls and was restored by IBTX, but not by TRAM-34/apamin. K(+) channel blockers were without effect on EC basal E(m) in controls. Similarly, IBTX blocked acetylcholine-induced dilation in arteries from CH rats, but was without effect in controls, whereas TRAM-34/apamin eliminated dilation in controls. Acetylcholine-induced EC hyperpolarization and calcium responses were inhibited by IBTX in CH arteries and by TRAM-34/apamin in controls. Patch-clamp experiments on freshly isolated ECs demonstrated greater K(+) current in cells from CH rats that was normalized by IBTX. IBTX was without effect on K(+) current in controls. We conclude that hypobaric CH induces increased endothelial BK(Ca) channel activity that contributes to reduced myogenic responsiveness and EC and VSM cell hyperpolarization.

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels is present following in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. We tested the hypothesis that CH removes an inhibitory effect of the scaffolding domain of caveolin-1 (Cav-1) on EC BK(Ca) channels to permit activation, thereby affecting vasoreactivity. Experiments were performed on gracilis resistance arteries and ECs from control and CH-exposed (380 mmHg barometric pressure for 48 h) rats. EC membrane potential was hyperpolarized in arteries from CH-exposed rats and arteries treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD) compared with controls. Hyperpolarization was reversed by the BK(Ca) channel antagonist iberiotoxin (IBTX) or by a scaffolding domain peptide of Cav-1 (AP-CAV). Patch-clamp experiments documented an IBTX-sensitive current in ECs from CH-exposed rats and in MBCD-treated cells that was not present in controls. This current was enhanced by the BK(Ca) channel activator NS-1619 and blocked by AP-CAV or cholesterol supplementation. EC BK(Ca) channels displayed similar unitary conductance but greater Ca(2+) sensitivity than BK(Ca) channels from vascular smooth muscle. Immunofluorescence imaging demonstrated greater association of BK(Ca) α-subunits with Cav-1 in control arteries than in arteries from CH-exposed rats, although fluorescence intensity for each protein did not differ between groups. Finally, AP-CAV restored myogenic and phenylephrine-induced constriction in arteries from CH-exposed rats without affecting controls. AP-CAV similarly restored diminished reactivity to phenylephrine in control arteries pretreated with MBCD. We conclude that CH unmasks EC BK(Ca) channel activity by removing an inhibitory action of the Cav-1 scaffolding domain that may depend on cellular cholesterol levels.

elevated neopterin levels had IL-6-independent association with prevalent frailty, suggesting potential monocyte/macrophage-mediated immune activation in the frail elderly.

Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) stimulates proliferation of hematopoietic cells of the macrophage and granulocyte lineages and is used clinically to treat neutropenia and other myeloid disorders. Because of its short circulating half-life GM-CSF is administered to patients by daily injection. We describe here the engineering of highly potent, long acting human GM-CSF proteins through site-specific modification of GM-CSF cysteine analogs with a cysteine-reactive polyethylene glycol (PEG) reagent. Thirteen cysteine analogs of GM-CSF were constructed, primarily in non-helical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogs were properly processed, but insoluble following secretion into the Escherichia coli periplasm. The proteins were refolded and purified by column chromatography. Ten of the cysteine analogs could be modified with a 5 kDa-maleimide PEG and seven of the mono-PEGylated proteins were purified by ion-exchange column chromatography. Biological activities of the 13 cysteine analogs and seven PEGylated cysteine analogs were comparable to that of wild type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analog was modified with larger 10 kDa-, 20 kDa-and 40 kDaPEGs, with only minimal loss of in vitro bioactivity. Pharmacokinetic experiments in rats demonstrated that the PEGylated proteins had up to 47-fold longer circulating half-lives than wild type GM-CSF. These data demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting GM-CSF analogs, and provide further evidence that the non-helical regions of human GM-CSF examined are largely non-essential for biological activity of the protein.

When subject to a transmural pressure gradient resistance arteries develop a spontaneous, intrinsically initiated contraction which varies according to the pressure stimulus and occurs in the absence of vasoconstrictor agonists. Such pressure-dependent active changes in vascular tone are indicative of the vascular myogenic response and contribute to autoregulation and the setting of total peripheral resistance and hence blood pressure regulation. The myogenic behaviour of blood vessels provides the background tone upon which other vasomotor influences act. Hypertension is associated with a raised vascular resistance and in this article the evidence for increased myogenic activity contributing to the raised vascular resistance is reviewed. Although there are some cases that provide evidence for exaggerated myogenic responsiveness in resistance arteries taken from hypertensive animals it is not possible to conclude that enhanced myogenic contractile responses within normal pressure ranges contribute to the raised total peripheral resistance. However, the myogenic tone of the resistance arteries of the various vascular beds is subject to differing modulatory influences in hypertensive animals and their normotensive controls which may contribute to the aetiology of hypertension. Experimental Physiology (2002) 87.5, 527-534. 2399