Osteoarthritis as a Systemic Disease Promoted Prostate Cancer In Vivo and In Vitro.

Osteoarthritis (OA) is increasing worldwide, and previous work found that OA increases systemic cartilage oligomeric matrix protein (COMP), which has also been implicated in prostate cancer (PCa). As such, we sought to investigate whether OA augments PCa progression. Cellular proliferation and migration of RM1 murine PCa cells treated with interleukin (IL)-1α, COMP, IL-1α + COMP, or conditioned media from cartilage explants treated with IL-1α (representing OA media) and with inhibitors of COMP were assessed. A validated murine model was used for tumor growth and marker expression analysis. Both proliferation and migration were greater in PCa cells treated with OA media compared to controls (p < 0.001), which was not seen with direct application of the stimulants. Migration and proliferation were not negatively affected when OA media was mixed with downstream and COMP inhibitors compared to controls (p > 0.05 for all). Mice with OA developed tumors 100% of the time, whereas mice without OA only 83.4% (p = 0.478). Tumor weight correlated with OA severity (Pearson correlation = 0.813, p = 0.002). Moreover, tumors from mice with OA demonstrated increased Ki-67 expression compared to controls (mean 24.56% vs. 6.91%, p = 0.004) but no difference in CD31, PSMA, or COMP expression (p > 0.05). OA appears to promote prostate cancer in vitro and in vivo.

Angiotensin-(1-7) reduces doxorubicin-induced cardiac dysfunction in male and female Sprague-Dawley rats through antioxidant mechanisms.

Doxorubicin (Dox) is an effective chemotherapeutic for a variety of pediatric malignancies. Unfortunately, Dox administration often results in a cumulative dose-dependent cardiotoxicity that manifests with marked oxidative stress, leading to heart failure. Adjunct therapies are needed to mitigate Dox cardiotoxicity and enhance quality of life in pediatric patients with cancer. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous hormone with cardioprotective properties. This study investigated whether adjunct Ang-(1-7) attenuates cardiotoxicity resulting from exposure to Dox in male and female juvenile rats. Dox significantly reduced body mass, and the addition of Ang-(1-7) had no effect. However, adjunct Ang-(1-7) prevented Dox-mediated diastolic dysfunction, including markers of decreased passive filling as measured by reduced early diastole mitral valve flow velocity peak (E) (P < 0.05) and early diastole mitral valve annulus peak velocity (e'; P < 0.001) and increased E/e' (P < 0.001), an echocardiographic measure of diastolic dysfunction. Since Dox treatment increases reactive oxygen species (ROS), the effect of Ang-(1-7) on oxidative by-products and enzymes that generate or reduce ROS was investigated. In hearts of male and female juvenile rats, Dox increased NADPH oxidase 4 (P < 0.05), a major cardiovascular NADPH oxidase isozyme that generates ROS, as well as 4-hydroxynonenal (P < 0.001) and malondialdehyde (P < 0.001), markers of lipid peroxidation; Ang-(1-7) prevented these effects of Dox. Cotreatment with Dox and Ang-(1-7) increased the antioxidant enzymes SOD1 (male: P < 0.05; female: P < 0.01) and catalase (P < 0.05), which likely contributed to reduced ROS. These results demonstrate that Ang-(1-7) prevents diastolic dysfunction in association with a reduction in ROS, suggesting that the heptapeptide hormone may serve as an effective adjuvant to improve Dox-induced cardiotoxicity.NEW & NOTEWORTHY Ang-(1-7) is a clinically safe peptide hormone with cardioprotective and antineoplastic properties that could be used as an adjuvant therapy to improve cancer treatment and mitigate the long-term cardiotoxicity associated with doxorubicin in pediatric patients with cancer.

Stabilization of Angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid.

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer.

Angiotensin-(1-7) prevents angiotensin II-induced fibrosis in cremaster microvessels.

OBJECTIVE: The effect of the heptapeptide hormone Ang-(1-7) on microvascular fibrosis in rats with Ang II-induced hypertension was investigated, since vascular fibrosis/remodeling plays a prominent role in hypertension-induced end-organ damage and Ang-(1-7) inhibits vascular growth and fibrosis. METHODS: Fibrosis of cremaster microvessels was studied in male Lewis rats infused with Ang II and/or Ang-(1-7). RESULTS: Ang II elevated systolic blood pressure by approximately 40 mmHg, while blood pressure was not changed by Ang-(1-7). Ang II increased perivascular fibrosis surrounding 20-50 µm arterioles as well as interstitial fibrosis; coadministration of Ang-(1-7) prevented the increases in fibrosis. The fibrotic factor CTGF and phospho-Smad 2/3, which upregulates CTGF, were increased by Ang II; this effect was prevented by coadministration of Ang-(1-7). Although TGF-ß phosphorylates Smad 2/3, TGF-ß was no different among treatment groups. In contrast, Ang II increased the MAP kinase phospho-ERK1/2, which also phosphorylates Smad; p-ERK was reduced by Ang-(1-7). Ang-(1-7), in the presence or absence of Ang II, upregulated the MAP kinase phosphatase DUSP1. CONCLUSIONS: These results suggest that Ang-(1-7) increases DUSP1 to reduce MAP kinase/Smad/CTGF signaling and decrease fibrosis in resistance arterioles, to attenuate end-organ damage associated with chronic hypertension.

Angiotensin-(1-7) reduces proliferation and angiogenesis of human prostate cancer xenografts with a decrease in angiogenic factors and an increase in sFlt-1.

BACKGROUND: Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. The purpose of this study was to determine the anti-proliferative and anti-angiogenic efficacy of angiotensin-(1-7) [Ang-(1-7)], an endogenous peptide hormone, in human prostate cancer xenografts. METHODS: Human LNCaP prostate cancer cells were injected into the flank of athymic mice and tumors were treated with Ang-(1-7) for 54 days. Tumor growth and angiogenesis were determined by immunohistochemistry and western blot hybridization. RESULTS: Ang-(1-7) markedly reduced the volume and wet weight of LNCaP xenograft tumors. Histological analysis of tumor sections from saline-treated mice showed increased Ki67 immunoreactivity and enhanced phosphorylation of the MAP kinases ERK1/2 compared to tumors from Ang-(1-7)-treated mice, suggesting that the heptapeptide reduces cell proliferation. Intratumoral vessel density was decreased in Ang-(1-7)-treated mice with an associated reduction in vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), suggesting that the heptapeptide attenuates vascularization by reducing angiogenic factors. Ang-(1-7) administration markedly increased the soluble fraction of VEGF receptor 1 (sFlt-1), with a concomitant reduction in VEGF receptors 1 and 2. sFlt-1 serves as a decoy receptor that traps VEGF and PlGF, making the ligands unavailable to membrane-bound VEGF receptors and preventing activation of pro-angiogenic signaling. CONCLUSIONS: The decrease in PlGF and VEGF coupled with the increase in sFlt-1 suggests that Ang-(1-7) may serve as a novel anti-angiogenic therapy for prostate cancer. Further, the pleiotropic mechanisms of action by Ang-(1-7) may limit angiogenic resistance that occurs with VEGF inhibitors or receptor blockers.

Angiotensin-(1-7) attenuates metastatic prostate cancer and reduces osteoclastogenesis.

BACKGROUND: Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone with anti-proliferative and anti-angiogenic properties. The primary objective of this study was to determine whether Ang-(1-7) effectively reduces prostate cancer metastasis in mice. METHODS: Human PC3 prostate cancer cells were injected into the aortic arch via the carotid artery of SCID mice pre-treated with Ang-(1-7) or injected into the tibia of athymic mice, administered Ang-(1-7) for 5 weeks beginning 2 weeks post-injection. Tumor growth and volume were determined by bioluminescent and magnetic resonance imaging. The presence of tumors was confirmed by hematoxylin and eosin staining; TRAP histochemistry was used to identify osteolytic lesions. The effect of Ang-(1-7) on osteoclastogenesis was assessed in differentiated bone marrow cells. RESULTS: Pre-treatment with Ang-(1-7) prevented metastatic tumor formation following intra-aortic injection of PC3 cells, while 83% of untreated mice developed tumors in metastatic sites. Circulating VEGF was significantly higher in control mice compared to mice administered Ang-(1-7). A 5-week regimen of the heptapeptide hormone attenuated intra-tibial tumor growth; Ang-(1-7) was significantly higher in the tibia of treated mice than in control animals. Osteoclastogenesis was reduced by 50% in bone marrow cells differentiated in the presence of Ang-(1-7), suggesting that the heptapeptide hormone prevents the formation of osteolytic lesions to reduce tumor survival in the bone microenvironment. CONCLUSIONS: These findings suggest that Ang-(1-7) may serve as an anti-angiogenic and anti-metastatic agent for advanced prostate cancer. By extension, the heptapeptide hormone may provide effective therapy for bone metastasis produced from primary tumors of the lung and breast.

Probiotic and Muscadine Grape Extract Interventions Shift the Gut Microbiome and Improve Metabolic Parameters in Female C57BL/6 Mice.

Obesity and Western-like diet consumption leads to gut microbiome dysbiosis, which is associated with the development of cardio-metabolic diseases and poor health outcomes. The objective of this study was to reduce Western diet-mediated gut microbial dysbiosis, metabolic dysfunction, and systemic inflammation through the administration of a novel combined intervention strategy (oral probiotic bacteria supplements and muscadine grape extract (MGE)). To do so, adult female C57BL/6 mice were fed a low-fat control or Western-style diet and sub-grouped into diet alone, probiotic intervention, antibiotic treatments, MGE supplementation, a combination of MGE and probiotics, or MGE and antibiotics for 13 weeks. Mouse body weight, visceral adipose tissue (VAT), liver, and mammary glands (MG) were weighed at the end of the study. Fecal 16S rRNA sequencing was performed to determine gut bacterial microbiome populations. Collagen, macrophage, and monocyte chemoattractant protein-1 (MCP-1) in the VAT and MG tissue were examined by immunohistochemistry. Adipocyte diameter was measured in VAT. Immunohistochemistry of intestinal segments was used to examine villi length, muscularis thickness, and goblet cell numbers. We show that dietary interventions in Western diet-fed mice modulated % body weight gain, visceral adiposity, MG weight, gut microbial populations, and inflammation. Intervention strategies in both diets effectively reduced VAT and MG fibrosis, VAT and MG macrophages, adipocyte diameter, and VAT and MG MCP-1. Interventions also improved intestinal health parameters. In conclusion, dietary intervention with MGE and probiotics modulates several microbial, inflammatory, and metabolic factors reducing poor health outcomes associated with Western diet intake.

Study design and methods for the pilot study of muscadine grape extract supplement to improve fatigue among older adult cancer survivors (FOCUS) trial.

INTRODUCTION: Fatigue is a prevalent symptom among both cancer survivors and older adults. Negative consequences of fatigue include increased sedentary behavior, decreased physical activity and function, and lower quality of life. Few pharmacologic interventions improve fatigue. Our preclinical and clinical data show promising effects of a muscadine grape extract supplement (MGES) on oxidative stress, mitochondrial bioenergetics, the microbiome, and the symptom of fatigue. This pilot study seeks to translate these observations to cancer survivorship by testing the preliminary effect of MGE supplementation on older adult cancer survivors with self-reported fatigue. MATERIALS AND METHODS: We designed a double-blinded placebo-controlled pilot study to evaluate preliminary efficacy of MGE supplementation versus placebo on fatigue among older adult cancer survivors (aged ≥65 years) who report baseline fatigue. Sixty-four participants will be enrolled and randomized 1:1 to twice daily MGES (four tablets twice daily) versus placebo for 12 weeks. The primary outcome is change in Patient-Reported Outcomes Measurement Information System (PROMIS) Fatigue score from baseline to 12 weeks. Secondary outcomes are change in self-reported physical function, physical fitness (6-min walk test), self-reported physical activity, global quality of life (QOL), and the Fried frailty index. Correlative biomarker assays will assess changes in 8-hydroxy-2 deoxyguanosine, peripheral blood mitochondrial function, inflammatory markers, and the gut microbiome. DISCUSSION: This pilot study builds on preclinical and clinical observations to estimate effects of MGE supplementation on fatigue, physical function, QOL, and biologic correlates in older adult cancer survivors. Trial registration #: CT.govNCT04495751; IND 152908.

Differences in oxidative stress status and expression of MKP-1 in dorsal medulla of transgenic rats with altered brain renin-angiotensin system.

ANG II-stimulated production of reactive oxygen species (ROS) through NADPH oxidase is suggested to activate MAPK pathways, which are implicated in neurally mediated pressor effects of ANG II. Emerging evidence suggests that ANG-(1-7) up regulates MAPK phosphatases to reduce MAPK signaling and attenuate actions of ANG II. Whether angiotensin peptides participate in long-term regulation of these systems in the brain is not known. Therefore, we determined tissue and mitochondrial ROS, as well as expression and activity of MAPK phosphatase-1 (MKP-1) in brain dorsal medullary tissue of hypertensive transgenic (mRen2)27 rats exhibiting higher ANG II/ANG-(1-7) tone or hypotensive transgenic rats with targeted decreased glial expression of angiotensinogen, ASrAOGEN (AS) exhibiting lower ANG II/ANG-(1-7) tone compared with normotensive Sprague-Dawley (SD) rats that serve as the control strain. Transgenic (mRen2)27 rats showed higher medullary tissue NADPH oxidase activity and dihydroethidium fluorescence in isolated mitochondria vs. SD or AS rats. Mitochondrial uncoupling protein 2 was lower in AS and unchanged in (mRen2)27 compared with SD rats. MKP-1 mRNA and protein expression were higher in AS and unchanged in (mRen2)27 compared with SD rats. AS rats also had lower phosphorylated ERK1/2 and JNK consistent with higher MKP-1 activity. Thus, an altered brain renin-angiotensin system influences oxidative stress status and regulates MKP-1 expression. However, there is a dissociation between these effects and the hemodynamic profiles. Higher ROS was associated with hypertension in (mRen2)27 and normal MKP-1, whereas the higher MKP-1 was associated with hypotension in AS, where ROS was normal relative to SD rats.

A Polyphenol-Rich Extract from Muscadine Grapes Prevents Hypertension-Induced Diastolic Dysfunction and Oxidative Stress.

Muscadine grapes are abundant in dietary polyphenols, but their effect on hypertension-induced cardiac damage is limited. This study assessed whether a muscadine grape skin/seed extract supplement (MGES) prevents hypertension-induced cardiac damage and oxidative stress. Male Sprague Dawley rats were treated for four weeks with drinking water, angiotensin II (Ang II) to induce hypertension, MGES, or both Ang II and MGES. Cardiac function assessed by echocardiography showed that Ang II increased systolic blood pressure while MGES alone or in combination with Ang II had no effect. Ang II increased E/e', an indicator of left ventricular filling pressure and diastolic dysfunction, by 41% compared to Control and co-treatment with MGES prevented the Ang II-mediated increase, suggesting that the extract attenuated hypertension-induced diastolic function. Ang II infusion increased urinary 8-hydroxy-2'-deoxyguanosine and cardiac 4-hydroxynonenal and malondialdehyde, which were prevented by the extract. The antioxidant enzymes catalase and superoxide dismutase 1 activity and mRNA were increased significantly in animals treated with MGES alone or in combination with Ang II, suggesting that the extract upregulates oxidative stress defense mechanisms in cardiac tissue. Thus, MGES may serve as a medical food to protect the heart from hypertension-induced diastolic dysfunction caused in part by excessive reactive oxygen species production.

Angiotensin-(1-7) reduces doxorubicin-induced aortic arch dysfunction in male and female juvenile Sprague Dawley rats through pleiotropic mechanisms.

Doxorubicin (Dox), an effective chemotherapeutic, can cause cumulative dose-dependent cardiovascular toxicity, which may manifest as vascular dysfunction leading to long-term end-organ damage. Currently, there are no effective treatments to mitigate Dox-induced vascular damage in cancer patients, particularly pediatric patients. We showed that angiotensin-(1-7) [Ang-(1-7)], an endogenous peptide hormone, mitigated cardiac damage in Dox-treated juvenile rats. In this study assessing aortic stiffness, juvenile male and female rats were administered a clinically equivalent dose of Dox (21-24 mg/kg) over 6 weeks, in the presence and absence of Ang-(1-7) [24 µg/kg/h]. Aortic function was measured using echocardiography. Ang-(1-7) reduced the Dox-mediated increase in pulse wave velocity, a measure of arterial stiffness (males: p < 0.05; females: p < 0.001) as compared in control animals. Dox decreased aortic lumen diameter (p < 0.0001) and increased wall thickness (p < 0.01) in males, which was attenuated by Ang-(1-7). In male but not female aortic arches, Dox increased media hypertrophy (p < 0.05) and reduced elastin content (p < 0.001), which were prevented by Ang-(1-7). Conversely, Dox increased fibrosis (p < 0.0001) in juvenile female rats, which was reduced by Ang-(1-7). Adjunct Ang-(1-7) prevented the Dox-induced increase in total cell and nuclear pERK1/2 in the aortic intima and media of male rats and nuclear pSMAD2 in the intimal and medial regions of the aortic arches of both sexes. These results demonstrate that Ang-(1-7) attenuated Dox-induced aortic dysfunction in both sexes of juvenile rats, albeit through different mechanisms, suggesting that Ang-(1-7) may serve as an effective adjuvant to ameliorate cardiovascular and long-term end-organ damage in pediatric patients produced by anthracyclines.

Targeted UHPLC-MS Analysis Reveals Disparate Polyphenol Composition and Concentration in Muscadine Grape Supplements with Proportional Antioxidant Activity.

Muscadine grape supplements (MGS) with high polyphenol content are a potential therapeutic option to combat oxidative stress; however, the precise identity and concentration of individual phenolics in commercially processed MGSs is not well defined. We probed for 17 phenolic compounds by ultra-high pressure liquid chromatography and mass spectroscopy from distinct lots of four commercially processed MGSs composed of MG seed and/or skin waste products. The total phenolic content (TPC) and antioxidant capacity were highest in a dried water-extract MGS as compared to three ground seed and/or skin products. The TPC was not different between MGS lots from individual companies and remained stable for 3 years without microbial contamination. The extract MGS had the highest concentration of epicatechin, ellagic acid, gallic acid, procyanidin B2, catechin and catechin gallate compared to the other supplements. Only ellagic acid and gallic acid were detected in all four MGSs, while catechin and catechin gallate were below detection in two supplements. Based on gram weight, only the extract MGS prevented the angiotensin II-induced increase in malondialdehyde and 4-hydroxynonenol in rat H9c2 cardiomyocytes as well as upregulated superoxide dismutase and catalase. This study demonstrates that commercial MGSs differ in phenolic composition and concentration, resulting in disparate antioxidant activity.

Phase I Study of Muscadine Grape Extract for Patients With Advanced Cancer.

OBJECTIVE: Preclinical studies with muscadine grape extract (MGE) show antitumor activity and decreased systemic inflammation. This phase I study (NCT02583269) assessed safety and tolerability of a proprietary MGE preparation in patients with advanced solid tumors. METHODS: Patients with metastatic or unresectable cancers who were progressing on standard therapies were assigned to MGE in a standard 3+3 design. Five dose levels were tested (320 to 1600 mg total phenolics/d). Safety and maximum-tolerated dose were assessed after 4 weeks. Patients were evaluated for response at 8 weeks and continued on MGE if clinically stable. Secondary outcomes were response, survival, adherence, fatigue, and quality of life (QOL). RESULTS: In total, 23 patients (lung, n=7; gastrointestinal, n=7; genitourinary, n=6; other, n=3) received MGE capsules by mouth twice daily. The cohort [median age 72 years, 48% Eastern Cooperative Oncology Group (ECOG) 2] was heavily pretreated. After 4 weeks on MGE, possibly attributable adverse events grade 2 or higher were fatigue (n=1), decreased lymphocyte count (n=1), and constipation (n=2), including 1 dose-limiting toxicity for grade 3 constipation. Maximum-tolerated dose was not reached. No partial responses were observed. Median time on therapy was 8 weeks, with 29% of patients treated beyond 16 weeks and a median overall survival of 7.2 months. QOL and fatigue levels were stable from baseline to 8 weeks. Higher MGE dose was correlated with improvement in self-reported physical well-being QOL at 8 weeks (r=0.6; P=0.04). CONCLUSIONS: MGE is safe and well-tolerated in heavily pretreated and older cancer patients.  The potential anticancer properties and the effects of MGE on physical well-being and QOL metrics will be evaluated in future studies.

A Polyphenol-Rich Extract From Muscadine Grapes Inhibits Triple-Negative Breast Tumor Growth.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that tends to affect young women and has a high propensity to metastasize. No targeted treatments are available for this type of breast cancer due to a lack of estrogen or progesterone receptors or overexpression of human epidermal growth factor receptor type 2 overexpression. Currently, patients have no therapeutic options once standard of care is complete, indicating a need for safe and effective therapies to slow or prevent the progression of TNBC to metastatic disease. Studies showed that isolated polyphenols or polyphenol-rich muscadine grape extracts polyphenols inhibit the proliferation of various cancer cells including breast cancer. A proprietary muscadine grape extract (MGE) was administered to nude mice with human MDA-MB-231 TNBC atumors for 4 weeks to determine the effect of the extract on tumor growth. MGE decreased tumor volume in association with a reduction in the proliferative markers Ki67 and cyclin D1. To determine the molecular mechanisms for the MGE-induced reduction in tumor growth, mouse 4T1, MDA-MB-231, or human BT-549 TNBC cells were treated with MGE, and various signaling pathways were investigated. MGE reduced c-Met, differentially abrogated ERK/MAPK and AKT signaling, and decreased a downstream targets of ERK/MAPK and AKT pathways, cyclin D1. Cyclin D1 reduction was associated with retinoblastoma activation and cell cycle arrest in MDA-MB-231 TNBC cells. MGE-regulated molecular signaling pathways were functionally associated with a dose-dependent reduction in cell proliferation. The pluripotency of MGE and high index of safety and tolerability suggest that the extract may serve as a therapeutic to reduce TNBC progression to metastatic disease.

Angiotensin-(1-7) inhibits growth of human lung adenocarcinoma xenografts in nude mice through a reduction in cyclooxygenase-2.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide of the renin-angiotensin system with vasodilator and antiproliferative properties. Our previous studies showed that Ang-(1-7) reduced serum-stimulated growth of human lung cancer cells in vitro through activation of a unique AT((1-7)) receptor. The current study investigates the effect of Ang-(1-7) on lung tumor growth in vivo, using a human lung tumor xenograft model. Athymic mice with tumors resulting from injection of A549 human lung cancer cells were treated for 28 days with either i.v. saline or Ang-(1-7), delivered by implanted osmotic mini-pumps. Treatment with Ang-(1-7) reduced tumor volume by 30% compared with the size before treatment; in contrast, tumor size in the saline-treated animals increased 2.5-fold. These results correlate with a reduction in the proliferation marker Ki67 in the Ang-(1-7)-infused tumors when compared with the saline-infused tumor tissues. Treatment with Ang-(1-7) significantly reduced cyclooxygenase-2 (COX-2) mRNA and protein in tumors of Ang-(1-7)-infused mice when compared with mice treated with saline as well as in the parent A549 human lung cancer cells in tissue culture. These results suggest that Ang-(1-7) may decrease COX-2 activity and proinflammatory prostaglandins to inhibit lung tumor growth. In contrast, the heptapeptide had no effect on COX-1 mRNA in xenograft tumors or A549 cells. Because Ang-(1-7), a peptide with antithrombotic properties, reduces growth through activation of a selective AT((1-7)) receptor, our results suggest that the heptapeptide represents a novel treatment for lung cancer by reducing COX-2.

Effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) has emerged as a novel regulator of cardiac function and arterial pressure by converting angiotensin II (Ang II) into the vasodilator and antitrophic heptapeptide, angiotensin-(1-7) [Ang-(1-7)]. As the only known human homolog of ACE, the demonstration that ACE2 is insensitive to blockade by ACE inhibitors prompted us to define the effect of ACE inhibition on the ACE2 gene. METHODS AND RESULTS: Blood pressure, cardiac rate, and plasma and cardiac tissue levels of Ang II and Ang-(1-7), together with cardiac ACE2, neprilysin, Ang II type 1 receptor (AT1), and mas receptor mRNAs, were measured in Lewis rats 12 days after continuous administration of vehicle, lisinopril, losartan, or both drugs combined in their drinking water. Equivalent decreases in blood pressure were obtained in rats given lisinopril or losartan alone or in combination. ACE inhibitor therapy caused a 1.8-fold increase in plasma Ang-(1-7), decreased plasma Ang II, and increased cardiac ACE2 mRNA but not cardiac ACE2 activity. Losartan increased plasma levels of both Ang II and Ang-(1-7), as well as cardiac ACE2 mRNA and cardiac ACE2 activity. Combination therapy duplicated the effects found in rats medicated with lisinopril, except that cardiac ACE2 mRNA fell to values found in vehicle-treated rats. Losartan treatment but not lisinopril increased cardiac tissue levels of Ang II and Ang-(1-7), whereas none of the treatments had an effect on cardiac neprilysin mRNA. CONCLUSIONS: Selective blockade of either Ang II synthesis or activity induced increases in cardiac ACE2 gene expression and cardiac ACE2 activity, whereas the combination of losartan and lisinopril was associated with elevated cardiac ACE2 activity but not cardiac ACE2 mRNA. Although the predominant effect of ACE inhibition may result from the combined effect of reduced Ang II formation and Ang-(1-7) metabolism, the antihypertensive action of AT1 antagonists may in part be due to increased Ang II metabolism by ACE2.

Programs to Recruit and Retain a More Diverse Workforce in Biomedical Sciences Research.

To improve overall healthcare and to reduce health disparities, efforts must focus on increasing the diversity of personnel trained in the biomedical sciences. Here, we describe the development, implementation, and relative outcomes of three pipeline training programs in biomedical sciences research designed to increase workforce diversity institutionally, regionally, and nationally. We report on their effectiveness in improving the recruitment and retention of underrepresented minorities with the long-term goal of remedying health inequities and disparities.

Angiotensin-(1-7) Attenuates Skeletal Muscle Fibrosis and Stiffening in a Mouse Model of Extremity Sarcoma Radiation Therapy.

BACKGROUND: Radiation-induced fibrosis (RIF) of musculoskeletal tissue is a common complication of radiation therapy for extremity soft-tissue sarcoma, with no standardized strategy for prevention and treatment. Angiotensin-(1-7) (Ang-[1-7]), a well-tolerated endogenous heptapeptide hormone with antitumor and antifibrotic properties, was tested as a radioprotectant for RIF and stiffening of irradiated muscles. METHODS: Male CD-1 mice were randomized to one of three treatment groups: control, simulated sarcoma radiation therapy to the gastrocnemius and soleus muscles, or radiation therapy along with continuous Ang-(1-7) delivery initiated three days before radiation therapy. The biologically equivalent dose of radiation (∼100.3 Gy) absorbed by normal musculature during the course of radiation therapy for extremity sarcoma was delivered by means of four dose fractions of 7.3 Gy over two weeks. Fibrosis (n = 5 per group) and mechanical properties (n = 4 to 6 per group) of the muscles were measured at six weeks and four months after radiation therapy, and the intramuscular concentration of the profibrotic cytokines transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) (n = 8 to 10 per group) were measured at six weeks. RESULTS: Interstitial (p < 0.01) and perivascular (p < 0.05) fibrosis increased significantly in the muscles treated with radiation therapy alone versus the nonirradiated controls at both six weeks (interstitial, +89%; perivascular, +112%) and four months (interstitial, +154%; perivascular, +88%). The muscles treated with radiation alone also exhibited increased tension (p < 0.01) versus nonirradiated controls at both six weeks (+779%) and four months (+1761%) when placed under 5% strain, and at four months (+1390%; p < 0.001) under 10% strain. At four months, muscle stiffness had increased in the mice treated with radiation therapy alone (+90%; p = 0.002) compared with nonirradiated controls. TGF-ß production was also greater in this group at six weeks (+37%; p = 0.06) versus control. Ang-(1-7) administration prevented RIF and stiffening, with no differences observed for any other outcome between those receiving radiation therapy with Ang-(1-7) and the nonirradiated controls. Likewise, Ang-(1-7) mitigated the increase in TGF-ß and CTGF concentration from radiation therapy. CONCLUSIONS: Ang-(1-7) attenuated RIF, stiffening, and production of profibrotic cytokines that were elevated in mouse skeletal muscles after simulated radiation therapy for extremity sarcoma. CLINICAL RELEVANCE: Ang-(1-7) may serve as a potential therapy for the prevention of RIF in patients who require radiation therapy as adjuvant treatment for soft-tissue sarcoma.

Angiotensin II stimulates arachidonic acid release from bone marrow stromal cells.

INTRODUCTION: Angiotensin II (Ang II) is recognised as a regulator of haematopoiesis, but its actions within the bone marrow are not fully understood. Support of haematopoiesis by bone marrow stromal cells (MSC) is dependent on factors that include arachidonic acid and macrophage colony stimulating factor (MCSF), both of which are increased by Ang II stimulation in other tissues. To further elucidate the mechanisms of Ang II-regulated haematopoiesis, we determined whether Ang II-stimulation alters arachidonic acid release and MCSF secretion from MSC. METHODS: Cynomolgus monkey MSC isolated from bone marrow aspirates and the human HS-5 stromal cell line were studied for Ang II-mediated arachidonic acid (AA) release, while secretion of MCSF in response to Ang II was studied in HS-5 cells. Cells were labelled overnight with 3H-AA and the release of 3H-AA was measured in culture medium following 20 minutes stimulation with Ang II, alone or in combination with the AT1- or AT2-receptor antagonists, losartan and PD 123319, respectively. MCSF secretion into culture medium was measured using an enzyme immunoassay following 24 hours of treatment with Ang II alone or in combination with losartan or PD 123319. Phorbol-myristate-acetate, known to stimulate release of AA and MCSF, was used as a positive control in both experiments. RESULTS: In response to Ang II, release of 3H-AA from monkey and human MSC was increased (p<0.05) to 147+/-4% and 124+/-3% of control, respectively. The AT1- and AT2-receptor antagonists, losartan and PD 123319, individually reduced Ang II-stimulated 3H-AA release. In contrast, Ang II had no effect on secretion of MCSF from HS-5 cells. CONCLUSIONS: These results provide mechanistic evidence for Ang II-mediated haematopoiesis through AA release that may, in part, explain Ang II-facilitated recovery of haematopoiesis in experimental myelosuppression and the anaemias associated with Ang II receptor blockade.

Angiotensin-(1-7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts.

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced (3)H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT((1-7)) receptor antagonist [d-Ala(7)]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.