Vasoconstrictor Effects of Trace Amine-Associated Receptor Agonists in the Isolated Perfused Rat Kidney.

INTRODUCTION: Endogenous trace amines such as tryptamine and 3-iodothyronamine (T1AM) are present in mammalian tissues at very low concentrations. They produce their actions by activating surface G protein-coupled receptors known as trace amine-associated receptors (TAARs). OBJECTIVE: The study was designed to investigate the possible vasoconstrictor effects of tryptamine, T1AM, and the selective TAAR1 agonist RO5263397 in isolated perfused rat kidney. METHODS: Renal vascular reactivity experiment using male Wistar Kyoto (WKY, n = 76) and spontaneously hypertensive rats (SHRs, n = 81) were used in this study. RESULTS: Tryptamine (1011-106 mole), T1AM (1011-106 mole), and RO5263397 (1011-106 mole) increased perfusion pressure in preparations from WKY rats and SHRs in a dose-dependent manner. EPPTB, a selective TAAR1 antagonist (10-6 M), significantly (p < 0.05) reduced agonist-induced increase in perfusion pressure in both WKY rats and SHRs, suggesting a role for TAAR1 activation in these responses. The vasoconstrictor responses in both groups were significantly reduced by L-type dihydropyridine calcium channel blocker, Rho-kinase, and protein kinase C (PKC) inhibitors suggesting the involvement of extracellular calcium and enhanced calcium sensitization. Reactive oxygen species (ROS) scavenger TEMPO significantly inhibited the agonist-induced increase in perfusion pressure in preparations from SHRs but not WKY. CONCLUSION: The TAARs enhanced agonist-induced increase in perfusion causing vasoconstriction in the kidney involves the influx of extracellular calcium, increased calcium sensitization, and ROS in SHRs animals only.

Attitude and Practice of Pharmacy Students Toward Traditional Herbal Medicines, Sudan.

Purpose: Traditional herbal medicines (THMs) are widely used in Sudan, but there is scarcity of research on pharmacy students' attitudes and utilization of THMs. This study aims to assess the attitudes and practices of pharmacy students toward THM. Methods: A descriptive cross-sectional study was conducted among randomly selected undergraduate pharmacy students at the University of Khartoum (Sudan). Data were collected using a validated self-administered questionnaire adapted from previous studies. A statistical package for the social sciences was used for data analysis. Results: Among the 264 respondents, the average age was 21.8 years, and 84.8% of them were females. Nearly 85% of respondents were residents of Khartoum. The overall respondent's attitudes towards THMs were positive, it ranged from high (19.7%), moderate (79.9%). About 87.8% of respondents are interested in learning more about THMs. In addition, 36.4%, and 50.4% of respondents strongly agreed or agreed that THMs should be officially licensed and controlled. A 17.8% and 63.3% of respondents, respectively, reported using traditional herbal remedies either always or occasionally. Supermarkets (35.2%) and friends/family (36.4%) were the main sources of THMs for the participants, and oral administration was the most used route (87.1%). More than 90% of respondents said they would suggest THMs to others, and the most common justifications for doing so were their accessibility (46.6%) and affordability (16.3%). The most widely used herbs were Mentha spicata, Zingiber officinale, Acacia nilotica, Hibiscus sabdariffa, and Syzygium aromaticum. Data revealed a significant association between attitude levels and respondents' gender (p=0.046), year of study (p=0.000), and residence (p=0.017). Conclusion: Most undergraduate pharmacy students had a positive attitude and utilized THMs. Development of effective educational initiatives, research, and regulations are recommended to ensure the appropriate utilization of traditional herbal remedies.

Correction: COVID-19 stressors and mental health problems amongst women who arrived as refugees and those born in Australia.

[This corrects the article DOI: 10.1371/journal.pgph.0002073.].

Correction: COVID-19 stressors and mental health problems amongst women who arrived as refugees and those born in Australia.

[This corrects the article DOI: 10.1371/journal.pgph.0002073.].

COVID-19 stressors and mental health problems amongst women who arrived as refugees and those born in Australia.

Women from refugee background residing in high income countries are at greater mental health risk during the COVID-19 pandemic given their higher baseline prevalence of mental disorders, trauma exposures and social adversities. During the COVID-19 pandemic we drew on data from wave-4 of the WATCH cohort study, collected between October 2019 and June 2021. We conducted a cross-sectional analysis to compare the prevalence of common mental disorders (CMDs) from the sample of 650 consecutively recruited women, 339 (52.2%) from the refugee-background who were resettled in Australia and 311 (48.8%) randomly and contemporaneously selected Australian born women. We assessed COVID-19 psychosocial stressors: 1. COVID-related material hardship and 2. COVID-related fear and stress. We examined for associations between scores on these two items and CMDs in each group respectively. Compared to Australian-born woman, women from refugee background recorded a significantly higher prevalence of Major Depressive Disorder (MDD) (19.8% vs 13.5%), PTSD (9.7% vs 5.1%), Separation Anxiety Disorder (SEPAD) (19.8% vs 13.5%) and Persistent Complicated Bereavement Disorder (PCBD) (6.5% vs 2.9%). In refugee women, associations were found between COVID-related material hardship and CMDs [MDD, Relative Risk (RR) = 1.39, 95%CI: 1.02-1.89, p = 0.02] as well as between COVID-related fear and stress and CMDs (MDD, RR = 1.74, 95%CI: 1.04-2.90, p = 0.02 p = 0.02). For Australian-born women, associations were more commonly found between CMDs and material hardship. Our study demonstrates that both women from refugee background and those born in Australia are experiencing significant rates of CMD during the pandemic and that material hardship is an associated factor. We found that women from refugee background are at greater risk for mental health problems and are more likely to report an association of those problems with fear and stress related to COVID_19. All women, and particularly those from refugee background, require urgent and specialised attention to their mental health and psychosocial problems during this pandemic.

Vasodilator Effect of Trace Amines, 3-Iodothyronamine, and RO5263397 in the Rat Perfused Kidney: Comparison with Tryptamine.

INTRODUCTION: Trace amine-associated receptors (TAARs) are a family of G protein-coupled receptors and are widely distributed in the body. Activation of TAAR1 by specific agonists can produce a variety of physiological effects centrally and peripherally. The objective of this study was to investigate the vasodilator effect of two selective TAAR1 agonists 3-iodothyronamine (T1AM) and RO5263397 in the isolated perfused rat kidney preparation. METHODS: Kidneys were isolated and perfused with Krebs' solution, gassed with 95% oxygen and 5% carbon dioxide, through the renal artery. RESULTS: In preparations pre-constricted with methoxamine (5 × 10-6m), T1AM (10-10 - 10-6 mol), RO5263397 (10-10 - 10-6 mol), and tryptamine (10-10 - 10-6 mol) produced dose-dependent vasodilator responses. EPPTB (1 × 10-6m), a selective TAAR1 antagonist, had no effect on vasodilator responses induced by these agonists. A higher concentration of EPPTB (3 × 10-5m) produced a sustained increase in perfusion pressure but did not affect vasodilator responses to tryptamine, T1AM, and RO5263397. Agonist-induced vasodilator responses were slightly reduced by the removal of the endothelium but were not affected by L-NAME (1 × 10-4m), an inhibitor of nitric oxide synthesis. The vasodilator responses were significantly reduced by inhibiting calcium-activated (tetraethylammonium, 1 × 10-3m) and voltage-activated (4-AP, 1 × 10-3m) potassium channels. Tryptamine-, T1AM-, and RO5263397-induced vasodilator responses were significantly reduced by BMY7378, a 5-HT1A receptor antagonist. CONCLUSION: It was concluded that vasodilator responses produced by the TAAR1 agonists, T1AM, RO5263397, and tryptamine, were not mediated via TAAR1 but were probably via activation of 5-HT1A receptors.

Hydrogen sulfide donor GYY4137 attenuates vascular complications in mesenteric bed of streptozotocin-induced diabetic rats.

Hydrogen sulfide (H2S) has been reported to have beneficial effects in different pathological conditions. OBJECTIVES: the effects of chronic treatment of diabetic rats with GYY4137 (slow releasing H2S donor) or NaHS (fast releasing H2S donor) on the reactivity of the mesenteric bed to vasoactive agonists and the changes in its downstream effectors, ERK1/2 and p38 MAP Kinase have been investigated. In addition, the levels of nitric oxide (NO) and H2S in all groups were measured. METHODS: diabetes was induced by a single intraperitoneal (ip) injection of streptozotocin (STZ; 55 mg/kg). Sprague Dawley (SD; n = 10-12/group) rats were randomly divided into six groups: control, STZ-induced diabetic rats, GYY4137-treated control, NaHS-treated control, GYY4137-treated diabetic, and NaHS-treated diabetic. After 28 days of treatment, rats were sacrificed and mesenteric beds were isolated for functional or biochemical studies. The vascular reactivity of the perfused mesenteric bed to norepinephrine, carbachol and sodium nitroprusside were determined by measurement of changes in perfusion pressure. Western blotting was performed to measure the protein expression of ERK1/2, p38, eNOS, and H2S biosynthesizing enzymes cystathionine-ß-synthase and cystathionine-γ-lyase. NO and H2S levels were measured in all groups in isolated mesenteric tissues or plasma. RESULTS: diabetes resulted in a significant increase in vasoconstrictor responses to norepinephrine (e.g., 129.6 ± 6.77 mmHg in diabetic vs 89.3 ± 8.48 mmHg in control at 10-7 dose), and carbachol-induced vasodilation was significantly reduced in diabetic mesenteric bed (e.g., 68.9 ± 4.8 mmHg in diabetic vs 90.6 ± 2.2 mmHg in control at 10-7 dose). Chronic treatment of the diabetic rats with GYY4137 resulted in a significant improvement in the response to norepinephrine (e.g., 86.66 ± 8.04 mmHg in GYY4137-treated diabetic vs 129.6 ± 6.77 mmHg in untreated diabetic at 10-7 dose) or carbachol (e.g., 84.90 ± 2.48 mmHg in GYY4137-treated diabetic vs 68.9 ± 4.8 mmHg in untreated diabetic at 10-7 dose). The biochemical studies showed a marked reduction of the protein expression of ERK and p38 and a significant upregulation of the expression of eNOS and H2S synthesizing enzymes after chronic treatment with GYY4137. Plasma levels of NO and H2S were significantly elevated after treatment with GYY4137. However, H2S production in the mesenteric bed showed a marginal elevation in diabetic tissues compared to controls. CONCLUSION: the results indicate that GYY4137 may be a novel therapeutic tool to prevent diabetes-associated vascular dysfunction.

Perceived Academic Stress, Causes, and Coping Strategies Among Undergraduate Pharmacy Students During the COVID-19 Pandemic.

BACKGROUND: Academic stress is a common problem among medical students, and the COVID-19 health crisis lockdown further worsened it. High academic stress has a negative impact on students learning and overall performance. OBJECTIVE: To assess perceived academic stress, causes, and coping strategies among undergraduate pharmacy students during the COVID-19 pandemic. METHODS: A descriptive cross-sectional study was conducted among undergraduate pharmacy students at the University of Khartoum. Data were collected from randomly selected participants using three validated self-administered questionnaires; perceived stress scale, study habits inventory, and mental health inventory. Data were analyzed using SPSS software, and descriptive statistics and chi-square were employed. RESULTS: The response rate in our study was 99.6% (251/252). About 87% of the participants were females. The majority of participants (92%) experience academic stress, with a mean score (24.99 ± 5.159), the level of academic stress ranging from low (4.3%), moderate (73.2%), to high (22.5%). Approximately 80% of the percipients reported academic stress during all exam times with a mean score (25.33 ± 4.976). The level of academic stress was significantly associated with participants' gender (P-value: 0.042), and living conditions (P-value: 0.001). The most common factors that were significantly associated with academic stress were difficulty in remembering all that is studied (66.7%, P=0.006) and worrying about the exams (54.1%, P=0.011). Moreover, the most frequent strategies used to cope with academic stress were praying (84.4%) and maintaining some control over the situation (61.9%). CONCLUSION: The study revealed a high prevalence of academic stress among percipients. Academic counseling, monitoring of mental status, and implementation of stress reduction programs are highly recommended.

Attitudes towards gender roles and prevalence of intimate partner violence perpetrated against pregnant and postnatal women: Differences between women immigrants from conflict-affected countries and women born in Australia.

BACKGROUND: The aim was to compare, for the first time in a large systematic study, women born in conflict-affected countries who immigrated to Australia with women born in Australia for attitudes towards gender roles and men's use of IPV and the actual prevalence of IPV. The study also examined if any associations remained across the two timepoints of pregnancy and postpartum. METHODS: Women were interviewed during their first visit to one of three Australian public hospital antenatal clinics and re-interviewed at home six months after giving birth. A total of 1111 women completed both interviews, 583 were born in conflict-affected countries and 528 born in Australia. Associations between attitudes towards gender roles and men's use of IPV, socio-demographic characteristics and reported actual experiences of IPV were examined using bivariate and multiple logistic regression analyses. RESULTS: Attitudes toward inequitable gender roles including those that condone men's use of IPV, and prevalence of IPV, were significantly higher (p<0.001) among women born in conflict-affected countries compared to Australia-born women. Women born in conflict-affected countries with the strongest held attitudes towards gender roles and men's use of IPV had an adjusted odds ratio (aOR) of 3.18 for IPV at baseline (95% CI 1.85-5.47) and an aOR of 1.83 for IPV at follow-up (95% CI 1.11-3.01). Women born in Australia with the strongest held attitudes towards gender roles and IPV had an aOR of 7.12 for IPV at baseline (95% CI 2.12-23.92) and an aOR of 10.59 for IPV at follow-up (95% CI 2.21-50.75). CONCLUSIONS: Our results underscore the need for IPV prevention strategies sensitively targeted to communities from conflict-affected countries, and for awareness among clinicians of gender role attitudes that may condone men's use of IPV, and the associated risk of IPV. The study supports the need for culturally informed national strategies to promote gender equality and to challenge practices and attitudes that condone men's violence in spousal relationships.

GYY4137 attenuates functional impairment of corpus cavernosum and reduces fibrosis in rats with STZ-induced diabetes by inhibiting the TGF-ß1/Smad/CTGF pathway.

Erectile dysfunction (ED) is a common diabetic complication. Recent evidence has illuminated the role of hydrogen sulfide (H2S) as a dynamic mediator of the erection process. H2S is a potent endogenous relaxant gas. It has been shown to relax human and animal penile tissue in vitro and induce erection in animals in vivo. The reported penile expression of H2S-synthesizing enzymes also supports the potential role of the endogenous L-cysteine/H2S pathway in penile homeostasis. Several pathological changes take place in the diabetic penile tissue, including inflammation, oxidative stress, neuropathy and fibrosis of the corpus cavernosum (CC), the major erectile structure of the penis. The present study is experimental and has been performed in the diabetic rat model. The study will investigate the role of H2S as a potential protective mediator against diabetes-induced structural and functional alterations in the CC by examining if it: (1) reduces corporal contraction and/or enhances corporal relaxation following pharmacological stimulation, (2) attenuates fibromuscular changes in diabetic CC, and (3) whether there is a link with H2S plasma/urine level and CC tissue generation, as well as studying the expression of some proteins in the transforming growth factor (TGF)-ß1-associated pathway. The major findings of the study reveal that- compared to the nondiabetic controls - the diabetic animals CC showed: (1) augmented contraction and attenuated relaxation in response to phenylephrine and carbachol, respectively, (2) marked fibromuscular degeneration with a significantly lower smooth muscle/collagen ratio and upregulation of TGF-ß-1/Smad/CTGF fibrosis signaling pathway, (3) reduced H2S plasma and urinary levels and cavernosal tissue generation. Chronic GYY4137 treatment prevented most of these pathological changes in diabetic CC, thus may be considered a potential new strategy for the prevention and/or treatment of diabetes-induced ED.

Chronic Treatment With Hydrogen Sulfide Donor GYY4137 Mitigates Microglial and Astrocyte Activation in the Spinal Cord of Streptozotocin-Induced Diabetic Rats.

Long-term diabetic patients suffer immensely from diabetic neuropathy. This study was designed to investigate the effects of hydrogen sulfide (H2S) on peripheral neuropathy, activation of microglia, astrocytes, and the cascade secretion of proinflammatory cytokines in the streptozotocin (STZ)-induced peripheral diabetic neuropathy rat model. STZ-induced diabetic rats were treated with the water-soluble, slow-releasing H2S donor GYY4137 (50 mg/kg; i.p.) daily for 4 weeks. Antiallodynic/antihyperalgesic activities were evaluated using different tests and histopathological changes and the expression of proinflammatory cytokines in the spinal cord were examined. GYY4137 treatment produced neuroprotective effects in the spinal cord of diabetic animals and modulated their sensory deficits. The treatment decreased allodynia (p < 0.05) and mechanical hyperalgesia (p < 0.01) and restored thermal hyperalgesia (p < 0.001) compared with diabetic rats. The treatment decreased the microglial response and increased astrocyte counts in spinal cord gray and white matter compared with untreated diabetic rats. Proinflammatory cytokines were reduced in the treated group compared with diabetic rats. These results suggest that H2S has a potentially ameliorative effect on the neuropathic pain through the control of astrocyte activation and microglia-mediated inflammation, which may be considered as a possible treatment of peripheral nerve hypersensitivity in diabetic patients.

Prevalence and Risk Factors of Major Depressive Disorder Among Women at Public Antenatal Clinics From Refugee, Conflict-Affected, and Australian-Born Backgrounds.

Importance: Pregnancy may increase the risk of depression among women who self-identify as refugees and have resettled in high-income countries. To our knowledge, no large systematic studies among women with refugee backgrounds in the antenatal period have been conducted. Objectives: To compare the prevalence of major depressive disorder (MDD), trauma exposure, and other psychosocial risk factors among women who identify as refugees, women from the same conflict-affected countries, and women from the host nation and to test whether self-identification as a refugee indicates greater likelihood of prevalence and risk. Design, Setting, and Participants: This cross-sectional study was undertaken in 3 public antenatal clinics in Sydney and Melbourne, Australia, between January 2015 and December 2016. Overall, 1335 women (685 consecutively enrolled from conflict-affected backgrounds and 650 randomly selected from the host nation) participated. Data analysis was undertaken between June and September 2018. Exposures: One-hour interviews covering mental health, intimate partner violence, and other social measures. Main Outcome and Measures: World Health Organization measure for intimate partner violence and the Mini-International Neuropsychiatric Interview from the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) for MDD. To make a diagnosis, 1 of 2 items relating to being consistently depressed for 2 weeks and 3 further symptoms that cause personal distress or psychosocial dysfunction were endorsed. Results: Overall, 1335 women (84.8% overall response rate), comprising 685 (51.3%) from conflict-affected countries (women self-identifying as refugees: 289 [42.2%]) and 650 (48.7%) from the host nation, participated. The mean (SD) age was 29.7 (5.4) years among women from conflict-affected backgrounds and 29.0 (5.5) years among women born in the host nation. Conflict-affected countries included Iraq (260 [38.0%]), Lebanon (125 [18.2%]), Sri Lanka (71 [10.4%]), and Sudan (66 [9.6%]). Women who identified as refugees reported higher exposure to 2 to 3 (67 [23.2%]) and 4 or more (19 [6.6%]) general traumatic events compared with women from the host nation (103 [15.8%] and 21 [3.2%], respectively). Women who identified as refugees also reported higher exposure to 1 (147 [50.9%]) and 2 or more (97 [33.6%]) refugee-related traumatic events compared with women from the host nation (86 [13.2%] and 20 [3.1%], respectively). Women who identified as refugees reported higher rates of psychological intimate partner violence than women born in the host nation (124 [42.9%] vs 133 [20.5%]; P < .001). Women who identified as refugees were less likely to identify 5 or more supportive family or friends compared with women born in the host nation (36 [12.5%] vs 297 [45.7%]; P < .001). A greater proportion of women who identified as refugees reported experiencing 3 or more financial stressors compared with women born in the host nation (65 [22.5%] vs 41 [6.3%]; P < .001). Women who identified as refugees had the highest prevalence of MDD (94 [32.5%]), followed by women from other conflict-affected backgrounds (78 [19.7%]), and women born in the host nation (94 [14.5%]). Conclusion and Relevance: Women identifying as refugees reported a higher prevalence of MDD and all the indicators of adversity related to that disorder. Even after risk factors were accounted for, refugee status was associated with risk of MDD. Assessing whether women attending an antenatal clinic self-identify as refugees may offer an important indicator of risk of MDD and a range of associated psychosocial adversities.

Chronic administration of nano-sized PAMAM dendrimers in vivo inhibits EGFR-ERK1/2-ROCK signaling pathway and attenuates diabetes-induced vascular remodeling and dysfunction.

We investigated whether chronic administration of nano-sized polyamidoamine (PAMAM) dendrimers can have beneficial effects on diabetes-induced vascular dysfunction by inhibiting the epidermal growth factor receptor (EGFR)-ERK1/2-Rho kinase (ROCK)-a pathway known to be critical in the development of diabetic vascular complications. Daily administration of naked PAMAMs for up to 4 weeks to streptozotocin-induced diabetic male Wistar rats inhibited EGFR-ERK1/2-ROCK signaling and improved diabetes-induced vascular remodeling and dysfunction in a dose, generation (G6 > G5) and surface chemistry-dependent manner (cationic > anionic > neutral). PAMAMs, AG1478 (a selective EGFR inhibitor), or anti-EGFR siRNA also inhibited vascular EGFR-ERK1/2-ROCK signaling in vitro. These data showed that naked PAMAM dendrimers have the propensity to modulate key (e.g. EGFR) cell signaling cascades with associated pharmacological consequences in vivo that are dependent on their physicochemical properties. Thus, PAMAMs, alone or in combination with vasculoprotective agents, may have a beneficial role in the potential treatment of diabetes-induced vascular complications.

Alleviation of impaired reactivity in the corpus cavernosum of STZ-diabetic rats by slow-release H2S donor GYY4137.

GYY4137 is a novel hydrogen sulfide (H2S) releasing molecule with vasodilator activity. The objectives of this study were to investigate: (1) the pharmacological effect of GYY4137 on the reactivity of the corpus cavernosum (CC) from normal and diabetic rats; (2) the contribution of ATP-sensitive potassium (K-ATP) channels and nitric oxide (NO) pathway; (3) the reactivity to vasoactive agonists following ex vivo incubation of the diabetic rat CC with GYY4137. Longitudinal strips of CC from control and diabetic male Sprague-Dawley (SD) rats (n = 5-6 animals per group) were suspended in organ-baths. Responses to GYY4137, carbachol, or phenylephrine (PE) were determined by measurement of changes in isometric tension. The effects of acute incubation of the CC strips with L-NAME (NO synthase inhibitor) or glibenclamide (K-ATP channel inhibitor) on the relaxant responses to GYY4137 were examined. The effect of ex vivo incubation with GYY4137 (10-5 M) on the responses of CC to carbachol or PE was evaluated. We found that GYY4137 provoked relaxation in the CC strips, which was significantly reduced in the presence of L-NAME or glibenclamide. Ex vivo incubation of diabetic CC with GYY4137 resulted in a significant improvement in the vascular responses to the added agonists. We conclude that GYY4137 is a relaxant agonist in SD rats CC, and the response is mediated, at least in part, by NO and K-ATP channels. Brief incubation of diabetic CC with GYY4137 markedly improved the impaired vascular reactivity, thus raising the question whether chronic in vivo treatment of diabetic animals with GYY4137 would have any protective effect, which is worth further investigation.

Angiotensin-(1-7) Downregulates Diabetes-Induced cGMP Phosphodiesterase Activation in Rat Corpus Cavernosum.

Molecular mechanisms of the beneficial effects of angiotensin-(1-7), Ang-(1-7), in diabetes-related complications, including erectile dysfunction, remain unclear. We examined the effect of diabetes and/or Ang-(1-7) treatment on vascular reactivity and cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) in corpus cavernosum. Male Wistar rats were grouped as (1) control, (2) diabetic (streptozotocin, STZ, treated), (3) control + Ang-(1-7), and (4) diabetic + Ang-(1-7). Following 3 weeks of Ang-(1-7) treatment subsequent to induction of diabetes, rats were sacrificed. Penile cavernosal tissue was isolated to measure vascular reactivity, PDE gene expression and activity, and levels of p38MAP kinase, nitrites, and cGMP. Carbachol-induced vasorelaxant response after preincubation of corpus cavernosum with PE was significantly attenuated in diabetic rats, and Ang-(1-7) markedly corrected the diabetes-induced impairment. Gene expression and activity of PDE and p38MAP kinase were significantly increased in cavernosal tissue of diabetic rats, and Ang-(1-7) markedly attenuated STZ-induced effects. Ang-(1-7) significantly increased the levels of nitrite and cGMP in cavernosal tissue of control and diabetic rats. Cavernosal tissue of diabetic rats had significantly reduced cGMP levels and Ang-(1-7) markedly prevented the STZ-induced cGMP depletion. This study demonstrates that attenuation of diabetes-induced PDE activity might be one of the key mechanisms in the beneficial effects of Ang-(1-7).

Angiotensin-(1-7)-dependent vasorelaxation of the renal artery exhibits unique angiotensin and bradykinin receptor selectivity.

Angiotensin-(1-7) [Ang-(1-7)] exhibits blood pressure lowering actions, inhibits cell growth, and reduces tissue inflammation and fibrosis which may functionally antagonize an activated Ang II-AT1 receptor axis. Since the vascular actions of Ang-(1-7) and the associated receptor/signaling pathways vary in different vascular beds, the current study established the vasorelaxant properties of the heptapeptide in the renal artery of male Wistar male rats. Ang-(1-7) produced an endothelium-dependent vasodilator relaxation of isolated renal artery segments pre-contracted by a sub-maximal concentration of phenylephrine (PE) (3×10-7M). Ang-(1-7) induced vasodilation of the rat renal artery with an ED50 of 3±1nM and a maximal response of 42±5% (N=10). The two antagonists (10-5M each) for the AT7/Mas receptor (MasR) [D-Pro7]-Ang-(1-7) and [D-Ala7]-Ang-(1-7) significantly reduced the maximal response to 12±1% and 18±3%, respectively. Surprisingly, the AT2R receptor antagonist PD123319, the AT1R antagonist losartan and B2R antagonist HOE140 (10-6M each) also significantly reduced Ang-(1-7)-induced relaxation to 12±2%, 22±3% and 14±7%, respectively. Removal of the endothelium or addition of the soluble guanylate cyclase (sGC) inhibitor ODQ (10-5M) essentially abolished the vasorelaxant response to Ang-(1-7) (10±4% and 10±2%, P <0.05). Finally, the NOS inhibitor LNAME (10-4M) reduced the response to 13±2% (p<0.05), but the cyclooxygenase inhibitor indomethacin failed to block the Ang-(1-7) response. We conclude that Ang-(1-7) exhibits potent vasorelaxant actions in the isolated renal artery that are dependent on an intact endothelium and the apparent stimulation of a NO-sGC pathway. Moreover, Ang-(1-7)-dependent vasorelaxation was sensitive to antagonists against the AT7/Mas, AT1, AT2 and B2 receptor subtypes.

Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor.

Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1-7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1-7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1-7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1-7). Ang-(1-7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1-7) that could be prevented by D-Pro7-Ang-(1-7) in VSMC. These data suggest that Ang-(1-7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications.

Cationic Polyamidoamine Dendrimers as Modulators of EGFR Signaling In Vitro and In Vivo.

Cationic polyamidoamine (PAMAM) dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented) generation (G) 6 PAMAM dendrimer, Superfect (SF), stimulated epidermal growth factor receptor (EGFR) tyrosine kinase signaling-an important signaling cascade that regulates cell growth, survival and apoptosis- in cultured human embryonic kidney (HEK 293) cells. Here, we firstly studied the in vitro effects of Polyfect (PF), a non-activated (intact) G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p) of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM dendrimers in nanomedicine.

The dual targeting of EGFR and ErbB2 with the inhibitor Lapatinib corrects high glucose-induced apoptosis and vascular dysfunction by opposing multiple diabetes-induced signaling changes.

The epidermal growth factor receptors, EGFR and EGFR2 (ErbB2), appear important mediators of diabetes-induced vascular dysfunction. We investigated whether targeted dual inhibition of EGFR and ErbB2 with Lapatinib would be effective in treating diabetes-induced vascular dysfunction in a rat model of type 1 diabetes. In streptozotocin-induced diabetes, chronic 4-week oral or acute, ex vivo, administration of Lapatinib prevented the development of vascular dysfunction as indicated by the attenuation of the hyper-reactivity of the diabetic mesenteric vascular bed (MVB) to norephinephrine without correcting hyperglycemia. Chronic in vivo or acute ex vivo Lapatinib treatment also significantly attenuated diabetes-induced increases in phosphorylation of EGFR, ErbB2, ERK1/2, AKT, ROCK2 and IkB-alpha as well as normalized the reduced levels of phosphorylated FOXO3A, and eNOS (Ser1177) in the diabetic MVB. Similar results were observed in vascular smooth muscle cells (VSMCs) cultured in high glucose (25 mM) treated with Lapatinib or small interfering RNA (siRNA) targeting the ErbB2 receptor. Lapatinib also prevented high glucose-induced apoptosis in VSMC. Thus, Lapatinib corrects hyperglycemia-induced apoptosis and vascular dysfunction with concomitant reversal of diabetes or high glucose-induced signaling changes in EGFR/ErbB2 and downstream signaling pathways implying that targeted dual inhibition of EGFR/ErbB2 might be an effective vasculoprotective treatment strategy in diabetic patients.

Chronic treatment with Ang-(1-7) reverses abnormal reactivity in the corpus cavernosum and normalizes diabetes-induced changes in the protein levels of ACE, ACE2, ROCK1, ROCK2 and omega-hydroxylase in a rat model of type 1 diabetes.

Angiotensin-(1-7) [Ang-(1-7)] may have beneficial effects in diabetes mellitus-induced erectile dysfunction (DMIED) but its molecular actions in the diabetic corpus cavernosum (CC) are not known. We characterized the effects of diabetes and/or chronic in vivo administration of Ang-(1-7) on vascular reactivity in the rat corpus cavernosum (CC) and on protein expression levels of potential downstream effectors of the renin-angiotensin-aldosterone system (RAAS) such as angiotensin-converting enzyme (ACE), ACE2, Rho kinases 1 and 2 (ROCK1 and ROCK2), and omega-hydroxylase, the cytochrome-P450 enzyme that metabolizes arachidonic acid to form the vasoconstrictor, 20-hydroxyeicosatetraenoic acid. Streptozotocin-treated rats were chronicically administered Ang-(1-7) with or without A779, a Mas receptor antagonist, during weeks 4 to 6 of diabetes. Ang-(1-7) reversed diabetes-induced abnormal reactivity to vasoactive agents (endothelin-1, phenylepherine, and carbachol) in the CC without correcting hyperglycemia. Six weeks of diabetes led to elevated ACE, ROCK1, ROCK 2, and omega-hydroxylase and a concomitant decrease in ACE2 protein expression levels that were normalized by Ang-(1-7) treatment but not upon coadministration of A779. These data are supportive of the notion that the beneficial effects of Ang-(1-7) in DMIED involve counterregulation of diabetes-induced changes in ACE, ACE2, Rho kinases, and omega-hydroxylase proteins in the diabetic CC via a Mas receptor-dependent mechanism.