Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension.

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.

Role of caveolin-1 in endothelial BKCa channel regulation of vasoreactivity.

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-ß-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.

Novel role of endothelial BKCa channels in altered vasoreactivity following hypoxia.

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.

Increased oxidant activity mediates vascular dysfunction in vibration injury.

Occupational exposure to hand-operated vibrating tools causes a spectrum of pathological changes in the vascular, neurological, and musculoskeletal systems described as the hand-arm vibration syndrome (HAVS). Experiments were performed to determine the effects of acute vibration on the function of digital arteries. Rats paws were exposed to a vibrating platform (4 h, 125 Hz, constant acceleration of 49 m/s(2) root mean squared), and digital artery function was assessed subsequently in vitro using a pressure myograph system. Constriction to phenylephrine or 5-hydroxytryptamine was reduced in digital arteries from vibrated paws. However, after endothelium denudation, constriction to the agonists was no longer impaired in vibrated arteries. Inhibition of nitric-oxide synthase (NOS) with N(omega)-nitro-l-arginine methyl ester (l-NAME) increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries and abolished the vibration-induced depression in constrictor responses. However, nitric oxide (NO) activity, determined using the NO-sensitive probe 4-amino-5-methylamino-2', 7'-difluorofluorescein, was reduced in vibrated compared with control arteries. Endogenous levels of reactive oxygen species (ROS), determined using the ROS-sensitive probe 5-(and 6)-chloromethyl-2',7'-dichlorodihydro-fluorescein, were increased in vibrated compared with control arteries. The increased ROS levels were abolished by L-NAME or by catalase, which degrades extracellular hydrogen peroxide. Catalase also increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries and abolished the vibration-induced depression in constrictor responses. The results suggest that acute vibration causes vascular dysfunction in digital arteries by increasing ROS levels, which is probably mediated by uncoupling of endothelial NOS. Therefore, therapeutic strategies to inhibit ROS or augment NO activity may be beneficial in HAVS.

Adenoviral delivery of interleukin-10 fails to attenuate experimental Lyme disease.

Production of interleukin-10 (IL-10) by C57BL/6 mice following infection with Borrelia burgdorferi has been proposed as a mechanism whereby resistance to the development of experimental Lyme arthritis is maintained. In the current study, we sought to determine the role of IL-10 during infection of arthritis- and carditis-susceptible C3H mice. Infection of C3H IL-10(-/-) mice led to increased joint swelling and arthritis severity scores over those of wild-type C3H mice. Measurement of B. burgdorferi numbers in joints or disseminated tissues indicated a more efficient clearance of spirochetes in the absence of IL-10, similar to that reported in C57BL/6 IL-10(-/-) mice. However, in contrast to previous in vitro work, infection of C3H IL-10(-/-) mice led to decreased in vivo expression of the cytokines KC, IL-1beta, IL-4, and IL-12p70 in the infected joints. Finally, adenoviral expression of IL-10 in the infected joints of C3H mice was unable to modulate the development of severe Lyme arthritis and had no effect on spirochete clearance or Borrelia-specific antibody production. Development of Lyme carditis appeared to be independent of modulation by IL-10. These results suggest that IL-10 limits the development of joint inflammation in both arthritis-resistant and -susceptible mouse strains infected with B. burgdorferi and that increased IL-10 production cannot rescue genetic susceptibility to development of pathology in this model.

Malignant melanoma of the horn base in a Pygora goat.

A case of malignant melanoma, originating at the base of the left horn, was diagnosed in a white 11-year-old Pygora doe. At necropsy, the goat had an ulcerated, black-pigmented, 2.5-cm mass at the base of the left horn. Firm masses diffusely black on the cut surface were present at the left horn base, subcutaneously over the frontal bones, and infiltrating into the frontal sinuses, the submandibular lymph node, and left parotid salivary gland. The left maxillary premolars and molars were loosened from the periodontia. Multiple black foci of metastasis were observed in the liver. Histologically, the masses consisted predominantly of moderately pleomorphic, polyhedral to spindle cells that contained variable amounts of dark brown intracytoplasmic pigment granules. The tumor cells were positive for Melan A by immunohistochemical staining.

Influence of experimental reduction of arterial media : lumen ratio on agonist-stimulated contractions in hypertension.

OBJECTIVE: This study investigated the relationship between the arterial media thickness : lumen diameter (M : L) ratio and arterial contractile responses in the spontaneously hypertensive rat (SHR) and its Wistar-Kyoto control (WKY) under isobaric conditions. METHODS: Femoral arteries with an experimentally reduced M : L ratio were produced in one hindlimb of both rat strains, by partial ligation of the ipsilateral iliac artery. Arterial structure and contractile responses were assessed in an arteriograph. Contractile responses of these vessels to the vasoconstrictor agonists noradrenaline and phenylephrine were determined after generation of spontaneous myogenic tone and compared with those from contralateral hindlimbs determined at both estimated in-vivo pressures and at 80 mmHg. Control SHR and WKY arterial responses were also compared. RESULTS: Under both pressure conditions, relaxed M : L ratios were significantly greater in SHR than in WKY arteries and were also significantly reduced in arteries distal to the ligature in both strains. In no comparison was a greater M : L associated with a greater agonist-stimulated contractile response. However, increased M : L were associated with narrower lumens across the concentration response relationships under some, but not all, conditions. CONCLUSION: An increased arterial M : L ratio does not effect an exaggerated contractile function in myogenically active resistance arteries in vitro. However, narrower lumen diameters associated with the increased M : L would provide a means for increased vascular resistance. These observations support the hypothesis that an increased M : L does not provide a means for a contractile response amplifier but may provide a means for a vascular resistance amplifier under some circumstances.

Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor.

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 analogues with a cysteine-reactive poly(ethylene glycol) (PEG) reagent. Thirteen cysteine analogues of GM-CSF were constructed, primarily in nonhelical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogues 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 analogues 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 analogues and 7 PEGylated cysteine analogues were comparable to that of wild-type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analogue was modified with larger 10-, 20-, and 40-kDa PEGs, 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 analogues and provide further evidence that the nonhelical regions of human GM-CSF examined are largely nonessential for biological activity of the protein.

Arterial myogenic properties of the spontaneously hypertensive rat.

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.

Influence of experimental reduction of media/lumen ratio on arterial myogenic properties of spontaneously hypertensive and Wistar-Kyoto rats.

In the present study, myogenic properties of femoral arteries from control hindlimbs and those distal to external iliac artery partial ligation of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were assessed. Arterial pressure was reduced distal to the ligature in both strains. Media thickness/lumen diameter (M/L) ratios of control (unligatured) SHRs were greater than in unligatured WKY rats and were reduced in arteries distal to the ligature (ligatured) within each strain. In none of the comparisons was a greater M/L ratio associated with greater maximal myogenic contractions, but increased M/L ratios were associated with a shift of myogenic activity to a higher pressure range in all comparisons. SHR ligatured arteries produced greater pressure-dependent contractile responses than WKY rat unligatured arteries, although arterial structures were not significantly different. Wall stress was similar in all arteries within the 60-120 mmHg pressure range with myogenic tone in spite of large differences in arterial structure. The utilization of arteries with experimentally altered structure provides further evidence that increased M/L ratios are not associated with greater peak pressure-dependent contractile responses and that arterial wall stress is maintained within a narrow range through an interaction between arterial wall geometry and smooth muscle contractile function.