Weekly doxorubicin increases coronary arteriolar wall and adventitial thickness.

BACKGROUND: Doxorubicin (DOX) is associated with premature cardiovascular events including myocardial infarction. This study was performed to determine if the weekly administration of DOX influenced coronary arteriolar medial and/or adventitial wall thickening. METHODS: Thirty-two male Sprague-Dawley rats aged 25.1± 2.4 weeks were randomly divided into three groups and received weekly intraperitoneal injections of normal saline (saline, n = 7), or low (1.5 mg/kg to 1.75 mg/kg, n = 14) or high (2.5 mg/kg, n = 11) doses of DOX. The animals were treated for 2-12 weeks, and euthanized at pre-specified intervals (2, 4, 7, or 10+ weeks) to obtain histopathologic assessments of coronary arteriolar lumen diameter, medial wall thickness, adventitial wall thickness, and total wall thickness (medial thickness + adventitial thickness). RESULTS: Lumen diameter was similar across all groups (saline: 315±34 µm, low DOX: 286±24 µm, high DOX: 242±27 µm; p = 0.22). In comparison to animals receiving weekly saline, animals receiving weekly injections of 2.5 mg/kg of DOX experienced an increase in medial (23±2 µm vs. 13±3 µm; p = 0.005), and total wall thickness (51±4 µm vs. 36±5 µm; p = 0.022), respectively. These increases, as well as adventitial thickening became more prominent after normalizing for lumen diameter (p<0.05 to p<0.001) and after adjusting for age, weight, and total cumulative DOX dose (p = 0.02 to p = 0.01). Animals receiving low dose DOX trended toward increases in adventitial and total wall thickness after normalization to lumen diameter and accounting for age, weight, and total cumulative DOX dose (p = 0.06 and 0.09, respectively). CONCLUSION: In conclusion, these data demonstrate that weekly treatment of rats with higher doses of DOX increases coronary arteriolar medial, adventitial, and total wall thickness. Future studies are warranted to determine if DOX related coronary arteriolar effects are reversible or preventable, exacerbate the known cardiomyopathic effects of DOX, influence altered resting or stress-induced myocardial perfusion, or contribute to the occurrence of myocardial infarction.

The effect of botulinum neurotoxin-A on blood flow in rats: a potential mechanism for treatment of Raynaud phenomenon.

PURPOSE: Botulinum neurotoxin-A (BoNTA) is used to treat several disorders, including Raynaud phenomenon. Recent investigations cite toxin-induced increases in blood flow, but no mechanism for BoNTA's actions is proposed. This study hypothesized that local application of BoNTA causes arteriolar vasodilation through sympathetic blockade and results in increased blood flow. METHODS: Microvascular effects of BoNTA were assessed using a rat cremaster preparation. Cremaster microvascular diameters were measured in the muscle before and after treatment with the muscle paralytic agent gallamine triethiodide. Preparations were then treated with one of the following: BoNTA (4, 6, or 10 units), BoNTA dilution vehicle, or denatured BoNTA. Arteriolar diameters were measured repeatedly over the observation period. Additional preparations were treated with either tetrodotoxin or prazosin and rauwolscine before BoNTA to confirm that the observed vasodilatory responses were the result of sympathetic neural inhibition. RESULTS: The BoNTA application resulted in a significant dose-dependent vasodilation (13% to 15%) of observed cremaster arterioles. Control treatments did not cause vasodilation. Both tetrodotoxin and prazosin/rauwolscine treatments elicited similar vasodilatory effects, with no additional vasodilation elicited by BoNTA. Addition of sodium nitroprusside following BoNTA elicited further vasodilation. In addition, systemic arterial pressure was unaffected by the local administration of BoNTA. CONCLUSIONS: Local application of BoNTA results in arteriolar dilation that yields an approximate 69% increase in blood flow, without changing systemic arterial pressure. A BoNTA-mediated vasodilation through sympathetic blockade is a likely mechanism to explain the increase in blood flow reported after treatment with the toxin. CLINICAL RELEVANCE: The ability of BoNTA to inhibit sympathetic nervous input reduces vasoconstriction, which is the most likely mechanism for improvement seen in Raynaud phenomenon patients following BoNTA injection.

PKC-dependent regulation of the receptor locus dominates functional consequences of cysteinyl leukotriene type 1 receptor activation.

Leukotrienes are important lipid mediators of asthma that contribute to airway inflammation and bronchoconstriction. Critical mechanisms for physiological regulation of the main G protein-coupled receptor (GPCR) mediating the leukotriene responses in asthma, cysteinyl leukotriene type 1 receptor (CysLT1R), have not been delineated. Although desensitization of GPCRs is a well-established phenomenon, studies demonstrating its physiological relevance are lacking. Here, we demonstrate that relief of PKC-mediated desensitization of endogenous CysLT1Rs augments multiple LTD4-stimulated cellular functions, with associated increases in intracellular signaling events. In analyses of airway smooth muscle contraction ex vivo, PKC inhibition augmented LTD4-stimulated contraction, and increased phosphoinositide hydrolysis and calcium flux in both murine and human airway smooth muscle cells. Similarly, for human monocytes, PKC inhibition augmented LTD4-stimulated calcium flux and cell migration assessed in transwell chamber experiments and also augmented LTD4-induced production of monocyte chemotactic protein assessed by ELISA. In contrast, PKC inhibition had no effect or slightly attenuated these cell functions and signaling events promoted by other receptor agonists, suggesting that despite antithetical effects on downstream events, desensitization of the CysLT1R is the principal mechanism by which PKC regulates the functional consequences of CysLT1R activation.

Nicotine upregulates the expression of P2Y12 on vascular cells and megakaryoblasts.

BACKGROUND: P2Y12 is the major platelet receptor that mediates ADP-induced aggregation. P2Y12 is also expressed by vascular cells. The factors that regulate P2Y12 expression have not been determined. Since nicotine (NIC) has effects on platelet activation and vascular function, and because nicotinic and purinerigic receptors may interact, we determined whether nicotine altered P2Y12 expression. METHODS: Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Immunoblotting for P2Y12, P2Y2, and actin was performed. RESULTS: Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 (but not P2Y2) expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. CONCLUSION: Nicotine induces the expression of P2Y12 in vascular cells and megakaryoblasts, and is mediated by nicotinic acetylcholine receptors. Smokers exhibit higher platelet P2Y12, possibly mediated via nicotine. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall. CONDENSED ABSTRACT: The factors that regulate the expression of P2Y12, the platelet ADP receptor, have not been determined. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall.

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling.

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.