Role for ovarian hormones in purinoceptor-dependent natriuresis.

BACKGROUND: Premenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats. METHODS: To test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750 µg/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1δ and ENaCα) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000 nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors. RESULTS: Acute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3. CONCLUSION: These data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Measuring Cutaneous Lesions: Trends in Clinical Practice.

BACKGROUND: Knowing the size of a cutaneous lesion can be important for tracking its progression over time, selecting the proper treatment modality, surgical planning, determining prognosis, and accurate billing. However, providers vary in their consistency, accuracy, and methods of measuring cutaneous lesions. OBJECTIVE: To investigate the clinical practices of US dermatologists and dermatologic surgeons regarding how they determine the size of cutaneous lesions. MATERIALS AND METHODS: A survey was electronically distributed to members of the American Society for Dermatologic Surgery. RESULTS: Four hundred twenty-six dermatologists completed the online survey. When a lesion is suspected to be malignant, 85% of respondents obtained exact measurements most, if not all, of the time; however, only 8% did for benign lesions. Most providers determined lesion sizes themselves rather than delegating to staff. When performing visual estimation, approximately three-quarters believed that they were accurate to within 1 to 2 mm. The top reasons for obtaining exact measurements were for tracking atypical pigmented lesions, determining treatment pathways, and accurate billing. CONCLUSION: The majority of respondents believed that lesion size affected management decisions; however, the need for exact measurement remains controversial, particularly for benign lesions. Future studies may investigate whether taking exact versus estimated measurements has an effect on outcomes.

Retention of Mohs surgeons in academic dermatology.

BACKGROUND: Retention of academic Mohs surgeons is important for the growth of this specialty and teaching of residents and students. OBJECTIVE: To examine factors that influence retention of Mohs surgeons in academics and to better understand reasons for their departure. MATERIALS AND METHODS: A survey was electronically distributed to academic Mohs surgeons in the American College of Mohs Surgery, asking them to rate the importance of several variables on their decision to remain in academia. Private practice Mohs surgeons who had left academics were also surveyed. RESULTS: Two hundred thirty-six dermatologic surgeons completed the survey. Twenty-nine percent work full time in academics, and approximately 7% work part time. The top reasons for practicing in the academic setting are intellectual stimulation, teaching opportunities, and collaboration with other university physicians and researchers. Seventy-one percent of respondents reported they would stay in academics, 7% indicated they would not, and 22% were unsure. Unfair compensation, inadequate support staff, poor leadership, increased bureaucracy, and decreased autonomy were top reasons that may compel a Mohs surgeon to leave. CONCLUSION: Opportunities for intellectual stimulation, collaboration, and teaching remain the main draw for academic Mohs surgeons. A supportive environment, strong leadership, and establishing fair compensation are imperative in ensuring their stay.

Clopidogrel preserves whole kidney autoregulatory behavior in ANG II-induced hypertension.

This study tested the hypothesis that P2Y12 receptor blockade with clopidogrel preserves renal autoregulatory ability during ANG II-induced hypertension. Clopidogrel was administered orally to male Sprague-Dawley rats chronically infused with ANG II. After 14 days of treatment, whole kidney autoregulation of renal blood flow was assessed in vivo in pentobarbital-anesthetized rats using an ultrasonic flow probe placed around the left renal artery. In ANG II-vehicle-treated rats, decreasing arterial pressure over a range from 160 to 100 mmHg resulted in a 25 ± 5% decrease in renal blood flow, demonstrating a significant loss of autoregulation with an autoregulatory index of 0.66 ± 0.15. However, clopidogrel treatment preserved autoregulatory behavior in ANG II-treated rats to levels indistinguishable from normotensive sham-operated (sham) rats (autoregulatory index: 0.04 ± 0.14). Compared with normotensive sham-vehicle-treated rats, ANG II infusion increased renal CD3-positive T cell infiltration by 66 ± 6%, induced significant thickening of the preglomerular vessels and glomerular basement membrane and increased glomerular collagen I deposition, tubulointerstitial fibrosis, damage to the proximal tubular brush border, and protein excretion. Clopidogrel significantly reduced renal infiltration of T cells by 39 ± 9% and prevented interstitial artery thickening, ANG II-induced damage to the glomerular basement membrane, deposition of collagen type I, and tubulointerstitial fibrosis, despite the maintenance of hypertension. These data demonstrate that systemic P2Y12 receptor blockade with clopidogrel protects against impairment of autoregulatory behavior and renal vascular injury in ANG II-induced hypertension, possibly by reducing renal T cell infiltration.

Increased Tc22 and Treg/CD8 ratio contribute to aggressive growth of transplant associated squamous cell carcinoma.

Immune suppressed organ transplant recipients suffer increased morbidity and mortality from primary cutaneous SCC. We studied tumor microenvironment in transplant-associated SCC (TSCC), immune-competent SCC and normal skin by IHC, IF and RT-PCR on surgical discard. We determined T cell polarization in TSCC and SCC by intracellular cytokine staining of T cell crawl outs from human skin explants. We studied the effects of IL-22, an inducer of keratinocyte proliferation, on SCC proliferation in vitro. SCC and TSCC are both associated with significantly higher numbers of CD3(+) and CD8(+) T cells compared to normal skin. TSCC showed a higher proportion of Foxp3(+) T regs to CD8(+) T cells compared to SCC and a lower percentage of IFN-γ producing CD4(+) T cells. TSCC, however, had a higher percentage of IL-22 producing CD8(+) T cells compared to SCC. TSCC showed more diffuse Ki67 and IL-22 receptor (IL-22R) expression by IHC. IL-22 induced SCC proliferation in vitro despite serum starvation. Diminished cytotoxic T cell function in TSCC due to decreased CD8/T-reg ratio may permit tumor progression. Increased IL-22 and IL-22R expression could accelerate tumor growth in transplant patients. IL-22 may be an attractive candidate for targeted therapy of SCC without endangering allograft survival.

CD200 upregulation in vascular endothelium surrounding cutaneous squamous cell carcinoma.

OBJECTIVE: To characterize the presence of CD200 and CD200 receptor (CD200R) in the human cutaneous squamous cell carcinoma (SCC) microenvironment and to define a possible role for the CD200 axis in immune evasion by SCC. DESIGN: Gene expression in SCC vs normal skin was studied. Laser capture microdissection was used to determine differential expression of CD200 in normal skin vs actinic keratosis vs SCC in situ vs invasive SCC. Immunofluorescence microscopy was used to define expression of CD200R on macrophages, myeloid dendritic cells, natural killer cells, and T cells in SCC vs normal skin. The effects of SCC supernatant on induction of CD200 in human blood endothelial cells was also examined. SETTING: Academic Medical Center with an established Section of Mohs and Dermatologic Surgery and an active Cutaneous Biology Research Program. PARTICIPANTS: Surgical discard tissue from deidentified patients and samples of normal skin from healthy volunteers were used in this study. MAIN OUTCOME MEASURES: Expression of CD200 on SCC-associated blood vessels; expression of CD200 receptor on SCC-associated macrophages and T cells; and induction of CD200 on endothelial cells by SCC supernatants. RESULTS: CD200 gene and message were upregulated in SCC stroma. Immunostaining revealed a higher number of CD200(+) cells in SCC stroma than in normal dermis (180.8 cells/mm(2) vs 24.6 cells/mm(2)) (P<.01). CD200 was further identified mainly on blood vessel endothelium in SCC. Tumor supernatant was able to induce CD200 expression on human dermal blood endothelial cells in culture. CD200R was identified on macrophages and dendritic cells in SCC microenvironment. CONCLUSIONS: CD200 expression on local blood vessels may promote tumor progression by suppressing CD200R myeloid cells during diapedesis. These data highlight a previously unrecognized mechanism of immune evasion by SCC and may provide guidance for the development of targeted therapy.

Immunosuppression preserves renal autoregulatory function and microvascular P2X(1) receptor reactivity in ANG II-hypertensive rats.

Autoregulation is critical for protecting the kidney against arterial pressure elevation and is compromised in some forms of hypertension. Evidence indicates that activated lymphocytes contribute importantly to cardiovascular injury in hypertension. We hypothesized that activated lymphocytes contribute to renal vascular dysfunction by impairing autoregulation and P2X(1) receptor signaling in ANG II-infused hypertensive rats. Male Sprague-Dawley rats receiving ANG II infusion were treated with a lymphocyte proliferation inhibitor, mycophenolate mofetil (MMF) for 2 wk. Autoregulation was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. ANG II-treated rats exhibited impaired autoregulation. At the single vessel level, pressure-mediated afferent arteriolar vasoconstriction was significantly blunted (P < 0.05 vs. control rats). At the whole kidney level, renal blood flow passively decreased as renal perfusion pressure was reduced. MMF treatment did not alter the ANG II-induced hypertensive state; however, MMF did preserve autoregulation. The autoregulatory profiles in both in vitro or in vivo settings were similar to the responses from control rats despite persistent hypertension. Autoregulatory responses are linked to P2X(1) receptor activation. Accordingly, afferent arteriolar responses to ATP and the P2X(1) receptor agonist ß,γ-methylene ATP were assessed. ATP- or ß,γ-methylene ATP-induced vasoconstriction was significantly attenuated in ANG II-infused hypertensive rats but was normalized by MMF treatment. Moreover, MMF prevented elevation of plasma transforming growth factor-ß1 concentration and lymphocyte and macrophage infiltration in ANG II-infused kidneys. These results suggest that anti-inflammatory treatment with MMF prevents lymphocyte infiltration and preserves autoregulation in ANG II-infused hypertensive rats, likely by normalizing P2X(1) receptor activation.

Langerhans cells from human cutaneous squamous cell carcinoma induce strong type 1 immunity.

Langerhans cells (LCs) are dendritic cells (DCs) localized to the epidermis. They should be the first antigen-presenting cells to encounter squamous cell carcinoma (SCC). The aim of this study was to investigate the ability of LCs isolated from human SCC to induce T-cell proliferation and polarization. We investigated the ability of LCs from SCC and peritumoral skin to induce T-cell proliferation and polarization. We also studied the effect of SCC supernatant on the ability of LCs from normal skin, in vitro-generated LCs, and DCs to activate and polarize T cells. LCs from SCC were stronger inducers of allogeneic CD4(+) and CD8(+) T-cell proliferation and IFN-γ production than LCs from peritumoral skin. We found that tumor supernatants (TSNs) were rich in immunosuppressive cytokines; despite this, allogeneic CD4(+) and CD8(+) T-cell proliferation and IFN-γ induction by LCs were augmented by TSN. Moreover, TSN facilitated IFN-γ induction by in vitro-generated LCs, but suppressed the ability of in vitro-generated DCs to expand allogeneic CD4(+) and CD8(+) T cells. We have demonstrated that LCs from SCC can induce type 1 immunity. TSN induces IFN-γ induction by in vitro-generated LCs. This contrasts greatly with prior studies showing that DCs from SCC cannot stimulate T cells. These data indicate that LCs may be superior to DCs for SCC immunotherapy and may provide a new rationale for harnessing LCs for the treatment of cancer patients.

Fluvastatin enhances sorafenib cytotoxicity in melanoma cells via modulation of AKT and JNK signaling pathways.

Most metastatic melanomas are refractory to current available therapy, underscoring the need to identify new effective treatments. Sorafenib, a multikinase inhibitor of the mitogen-activated protein kinase signaling pathway, showed promise in earlier stages of clinical development, but ultimately failed to demonstrate efficacy as a single agent in the treatment of melanoma. In order to enhance the efficacy of sorafenib in the treatment of melanoma, we tested over 2,000 naturally occurring compounds and marketed drugs in the presence of sorafenib in chemoresistant human melanoma cell lines also resistant to sorafenib-induced apoptosis. Of the 9 compounds identified as sorafenib sensitizers, we prioritized the cholesterol-lowering agent fluvastatin, based on its favorable pharmacokinetics and safety profile. Our results demonstrate that fluvastatin at 1 µM, a level safely achieved through oral administration, dramatically enhances the growth-inhibitory activity of clinically achievable concentrations of sorafenib in M14 and SKM-173 melanoma cells, with a 3.2- and 3.6-fold reduction in sorafenib 50% growth inhibition (GI50), respectively. Similar effects were observed for other melanoma cell lines. Combination indices analysis revealed a synergistic relationship between the two agents. Fluvastatin enhances sorafenib-mediated apoptosis as revealed through enhanced cleavage of poly (ADP-ribose) polymerase. In combination with sorafenib, fluvastatin treatment results in reduced levels of activated murine thymoma viral oncogene homolog along with enhanced levels of activated c-Jun N-terminal kinase. Sensitization to sorafenib is unique to fluvastatin, as other statins (pravastatin and simvastatin) do not enhance sorafenib-mediated growth inhibition. These promising results warrant further investigation into the clinical applicability of fluvastatin as an agent for enhancing the efficacy of sorafenib in the treatment of melanoma.

Clopidogrel, independent of the vascular P2Y12 receptor, improves arterial function in small mesenteric arteries from AngII-hypertensive rats.

The P2Y12 receptor antagonist clopidogrel blocks platelet aggregation, improves systemic endothelial nitric oxide bioavailability, and has anti-inflammatory effects. Since P2Y12 receptors have been identified in the vasculature, we hypothesized that clopidogrel ameliorates angiotensin II (Ang II) -induced vascular functional changes by blockade of P2Y12 receptors in the vasculature. Male Sprague Dawley rats were infused with Ang II (60 ng.min-1) or vehicle for 14 days. The animals were treated with clopidogrel (10mg*kg-1*day-1) or vehicle. Vascular reactivity was evaluated in second-order mesenteric arteries. Clopidogrel treatment did not change systolic blood pressure [(mmHg) control-vehicle, 117+/-7.1 vs. control- Clopidogrel, 125+/-4.2; AngII-vehicle, 197+/-10.7 vs. AngII-Clopidogrel, 198+/-5.2], but it normalized increased phenylephrine-induced vascular contractions [(%KCl) vehicle-treated, 182.2+/-18 vs. Clopidogrel, 133+/-14%), as well as impaired vasodilation to acetylcholine [(%) vehicle-treated, 71.7+/-2.2 vs. Clopidogrel, 85.3+/-2.8) in Ang II-treated animals. Vascular expression of P2Y12 receptor was determined by western blot. Pharmacological characterization of vascular P2Y12 was performed with the P2Y12 agonist 2-MeS-ADP. Although 2-MeSADP induced endothelium-dependent relaxation [(Emax %) = 71%+/-12), as well as contractile vascular responses (Emax %= 83+/-12) these actions are not mediated by P2Y12 receptor activation. 2-MeS-ADP produced similar vascular responses in control and Ang II rats. These results indicate potential effects of Clopidogrel, such as improvement of hypertension-related vascular functional changes that are not associated with direct actions of clopidogrel in the vasculature, supporting the concept that activated platelets contribute to endothelial dysfunction, possibly via impaired NO bioavailability.

P2 receptor regulation of [Ca2+]i in cultured mouse mesangial cells.

Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca(2+)](i)) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca(2+)](i) averaged 102 +/- 2 nM (n = 346). One hundred micromolar concentrations of ATP, ADP, 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), ATP-gamma-S, and UTP in normal Ca(2+) medium evoked peak increases in [Ca(2+)](i) of 866 +/- 111, 236 +/- 18, 316 +/- 26, 427 +/- 37, and 808 +/- 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca(2+)](i), whereas identical concentrations of adenosine, AMP, and alpha,beta-methylene ATP (alpha,beta-MeATP) had no detectable effect on [Ca(2+)](i). Removal of Ca(2+) from the extracellular medium had no significant effect on the peak increase in [Ca(2+)](i) induced by ATP, ADP, BzATP, ATP-gamma-S, or UTP compared with normal Ca(2+); however, Ca(2+)-free conditions did accelerate the rate of decline in [Ca(2+)](i) in cells treated with ATP and UTP. [Ca(2+)](i) was unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X(2), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. No evidence of P2X(1) and P2X(3) receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X(1), P2X(2), P2X(3), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca(2+)](i) by stimulating calcium influx from the extracellular medium and through mobilization of Ca(2+) from intracellular stores in cultured mouse mesangial cells.

Impaired Ca2+ signaling attenuates P2X receptor-mediated vasoconstriction of afferent arterioles in angiotensin II hypertension.

This study tested the hypothesis that afferent arteriolar responses to purinoceptor activation are attenuated, and Ca2+ signaling mechanisms are responsible for the blunted preglomerular vascular reactivity in angiotensin II (Ang II) hypertension. Experiments determined the effects of ATP, the P2X1 agonist beta,gamma-methylene ATP or the P2Y agonist UTP on arteriolar diameter using the juxtamedullary nephron technique and on renal myocyte intracellular Ca2+ concentration ([Ca2+]i) using single cell fluorescence microscopy. Six or 13 days of Ang II infusion significantly attenuated the vasoconstrictor responses to ATP and beta,gamma-methylene ATP (P<0.05). During exposure to ATP (1, 10, and 100 micromol/L), afferent diameter declined by 17+/-2%, 29+/-3%, and 30+/-2% in normal control rats and 8+/-3%, 7+/-3%, and 22+/-3% in kidneys of Ang II-infused rats (13 days). Renal myocyte intracellular calcium responses to ATP or beta,gamma-methylene ATP were also decreased in Ang II hypertensive rats. In myocytes of control rats, peak increases in [Ca2+]i averaged 107+/-21, 170+/-38, and 478+/-79 nmol/L at ATP concentrations of 1, 10, and 100 micromol/L, respectively. Ang II infusion for 13 days decreased the peak responses to ATP (1, 10, and 100 micromol/L) to 65+/-13, 102+/-20, and 367+/-73 nmol/L, respectively. The peak increases in [Ca2+]i in response to beta,gamma-methylene ATP were also reduced in Ang II hypertensive rats. However, angiotensin hypertension did not change the UTP-mediated vasoconstrictor responses or the myocyte calcium responses to UTP. These results indicate that the impaired autoregulatory response observed in Ang II-dependent hypertension can be attributed to impairment of P2X1 receptor-mediated signal transduction.