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Contains fulltext : 244306.pdf (Publisher’s version ) (Open Access) Patients are the stewards of their own care and hence their voice is important when designing and implementing research. Patients should be involved not only as participants in research that impacts their care, as the recipients of that care and any associated harms, but also as research collaborators in prioritising important questions from the patient perspective and designing the research and the ways in which is it most appropriate to involve patients. The PIONEER Consortium, an international multistakeholder collaboration lead by the European Association of Urology, has developed a core outcome set (COS) for localised and metastatic prostate cancer relevant to all stakeholders in particular patients. Throughout the work of PIONEER, patient representatives were involved as collaborators in setting the research agenda, and a wider group of patients was involved as participants in developing COSs, for instance in consensus meetings on choosing important outcomes and appropriate definitions. This publication showcases the process for COS development and highlights the most important recommendations to ultimately inform future research projects co-created between patients and other stakeholders. PATIENT SUMMARY: An important step in involving patients in the selection of outcomes for clinical trials, clinical audits, and real-world evidence is the development of a core outcome set (COS) that is relevant to all stakeholders. This report highlights the patient participation throughout our PIONEER COS development. TAKE HOME MESSAGE: An important step in involving patients in the selection of outcomes for clinical trials, clinical audits, and real-world evidence is to develop a core outcome set (COS) that is relevant to all stakeholders. As part of the work of the PIONEER Consortium, we aim to highlight the patient participation throughout our PIONEER COS development.

Background Urinary incontinence and sexual dysfunction are common after robot-assisted radical prostatectomy (RALP). New surgical techniques to improve these functions after the operation are under evaluation for example, preservation of endopelvic fascia during RALP. However, the benefits of this technique have not been critically scrutinized in a randomized setting. Aim In this study, we compared endopelvic fascia preserving operation with the standard surgical procedure in a randomized trial at the Tampere University Hospital, Finland. Methods A total of 158 men with localized prostate cancer and scheduled for RALP were randomized 1:1 into endopelvic fascia–preserving RALP or a control group that is, standard operation. All operations were performed by a single surgeon. Outcomes Urinary and sexual function were evaluated by the Expanded Prostate Cancer Index Composite-26 questionnaire at baseline and 3, 6, and 12 months after the surgery. Results There was no difference in urinary incontinence or sexual function between the groups at any time point (urinary incontinence domain at 12 months after RALP for fascia preserving and control group 73.6 ± 3 vs 78.9 ± 2.5 and sexual domain 43 ± 3.2 vs 40.3 ± 3, respectively). Clinical and pathologic tumor characteristics, duration of surgery, blood loss, rate of complications, and time to hospital discharge were similar between the study arms. Compliance of filling out the Expanded Prostate Cancer Index Composite-26 questionnaire varied from 91% to 98%, with no difference between study arms. Clinical Implications Based on our results, endopelvic fascia preservation alone during RALP is not recommended over the standard surgical method. Strengths & Limitations This is a randomized clinical study with sufficient statistical power. As a limitation, only a minority of participants underwent magnetic resonance imaging before the operation, thus we could not evaluate the role of urethral length or shape of the prostate. Urinary and sexual function results are based on questionnaires filled out by the patients, however, participants completed the surveys independently unassisted by health care personnel. Conclusion Endopelvic fascia–preserving RALP does not improve urinary continence or sexual function as compared with the standard surgical technique. Future studies aiming to improve functional outcomes after RALP should focus on evaluating other technique modifications. Siltari A, Riikonen J, Murtola TJ. Preservation of Endopelvic Fascia: Effects on Postoperative Incontinence and Sexual Function – A Randomized Clinical Trial. J Sex Med 2021;18:327–338.

IntroductionBlood cholesterol is likely a risk factor for prostate cancer prognosis and use of statins is associated with lowered risk of prostate cancer recurrence and progression. Furthermore, use of statins has been associated with prolonged time before development of castration resistance (CR) during androgen deprivation therapy (ADT) for prostate cancer. However, the efficacy of statins on delaying castration-resistance has not been tested in a randomised placebo-controlled setting.This study aims to test statins’ efficacy compared to placebo in delaying development of CR during ADT treatment for primary metastatic or recurrent prostate cancer. Secondary aim is to explore effect of statin intervention on prostate cancer mortality and lipid metabolism during ADT.Methods and analysisIn this randomised placebo-controlled trial, a total of 400 men with de novo metastatic prostate cancer or recurrent disease after primary treatment and starting ADT will be recruited and randomised 1:1 to use daily 80 mg of atorvastatin or placebo. All researchers, study nurses and patients will be blinded throughout the trial. Patients are followed until disease recurrence or death. Primary outcome is time to formation of CR after initiation of ADT. Serum lipid levels (total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and trigyserides) are analysed to test whether changes in serum cholesterol parameters during ADT predict length of treatment response. Furthermore, the trial will compare quality of life, cardiovascular morbidity, changes in blood glucose and circulating cell-free DNA, and urine lipidome during trial.Ethics and disseminationThis study is approved by the Regional ethics committees of the Pirkanmaa Hospital District, Science centre, Tampere, Finland (R18065M) and Tarto University Hospital, Tarto, Estonia (319/T-6). All participants read and sign informed consent form before study entry. After publication of results for the primary endpoints, anonymised summary metadata and statistical code will be made openly available. The data will not include any information that could make it possible to identify a given participant.Trial registration numberClinicaltrial.gov:NCT04026230, Eudra-CT: 2016-004774-17, protocol code: ESTO2, protocol date 10 September 2020 and version 6.

Statins have plausible biological effects against prostate cancer cells and are associated with improved disease-specific mortality. In current randomized placebo-controlled trial, low-dose atorvastatin caused no difference in relapses after radical prostatectomy in Asian men. Future trials should study higher statin doses at later disease stages with survival as the endpoint. See related article by Jeong et al., p. 5004

Contains fulltext : 291547.pdf (Publisher’s version ) (Open Access) OBJECTIVES: Genome-wide association studies have revealed over 200 genetic susceptibility loci for prostate cancer (PCa). By combining them, polygenic risk scores (PRS) can be generated to predict risk of PCa. We summarize the published evidence and conduct meta-analyses of PRS as a predictor of PCa risk in Caucasian men. PATIENTS AND METHODS: Data were extracted from 59 studies, with 16 studies including 17 separate analyses used in the main meta-analysis with a total of 20,786 cases and 69,106 controls identified through a systematic search of ten databases. Random effects meta-analysis was used to obtain pooled estimates of area under the receiver-operating characteristic curve (AUC). Meta-regression was used to assess the impact of number of single-nucleotide polymorphisms (SNPs) incorporated in PRS on AUC. Heterogeneity is expressed as I(2) scores. Publication bias was evaluated using funnel plots and Egger tests. RESULTS: The ability of PRS to identify men with PCa was modest (pooled AUC 0.63, 95% CI 0.62-0.64) with moderate consistency (I(2) 64%). Combining PRS with clinical variables increased the pooled AUC to 0.74 (0.68-0.81). Meta-regression showed only negligible increase in AUC for adding incremental SNPs. Despite moderate heterogeneity, publication bias was not evident. CONCLUSION: Typically, PRS accuracy is comparable to PSA or family history with a pooled AUC value 0.63 indicating mediocre performance for PRS alone. 01 april 2023

Modulation of lipid metabolism during cancer development and progression is one of the hallmarks of cancer in solid tumors; its importance in prostate cancer (PCa) has been demonstrated in numerous studies. Lipid metabolism is known to interact with androgen receptor signaling, an established driver of PCa progression and castration resistance. Similarly, immune cell infiltration into prostate tissue has been linked with the development and progression of PCa as well as with disturbances in lipid metabolism. Immuno-oncological drugs inhibit immune checkpoints to activate immune cells’ abilities to recognize and destroy cancer cells. These drugs have proved to be successful in treating some solid tumors, but in PCa their efficacy has been poor, with only a small minority of patients demonstrating a treatment response. In this review, we first describe the importance of lipid metabolism in PCa. Second, we collate current information on how modulation of lipid metabolism of cancer cells and the surrounding immune cells may impact the tumor’s immune responses which, in part, may explain the unimpressive results of immune-oncological treatments in PCa.

Dairy products are an important part of a nutritionally balanced diet as their constituents can affect the human state of health. By inhibiting the angiotensin I-converting enzyme, the tripeptides Val-Pro-Pro, Ile-Pro-Pro, and Leu-Pro-Pro can lower blood pressure. As these peptides are produced during fermentation, they are found in various dairy products like cheese, yoghurt, etc., but except for cheese only little is known about their content. To investigate how other dairy products contribute to a supply of these antihypertensive peptides, we developed and validated a fast and sensitive assay for quantification of the three tripeptides with LC-MS/MS combined with a simple protocol for extraction and SPE-purification from yoghurt, curd, or other products. Finally, the entire method was successfully applied to survey peptide concentrations in samples from local dairies and thus expands our awareness on the content of antihypertensive peptides in our food. (C) 2019 Elsevier Ltd. All rights reserved.

Epidemiological studies have revealed that consumption of milk and fermented dairy products is inversely associated with elevated blood pressure and with many of the risk factors of the metabolic syndrome. Previously, calcium was thought to be behind this phenomenon, but during the last 20 years, convincing evidence emerging from experimental, epidemiological and intervention studies has highlighted the important role of the small peptides formed during fermentation processes. This review provides an overview of the potential blood pressure lowering components present in dairy products with a special focus on casein‐derived tripeptides.

Introduction: Androgen-deprivation therapy (ADT) is standard in treatment of advanced prostate cancer (PCa). Common adverse effects include fatigue, weight gain, sarcopenia, and decreased libido leading to lowered quality of life (QoL). Supervised exercise improves overall QoL among PCa patients. Less is known, however, how it may affect metabolic adverse effects of ADT.The aim of the study was to explore the effect of exercise intervention on glucose and cholesterol levels, body composition, and to find out whether supervised exercise leads to greater physical activity in long-term compared to self-administered exercise in men with ADT. Materials and methods: Altogether, 45 men were recruited. One man withdrew the consent at baseline and 5 dropped out during the study. Thus, 39 men completed the study. Subjects were randomly assigned into either supervised exercise for 3 months (n=27) or control group of self-administered exercise (n=17). In the supervised group, participants attended 1.5-hour group exercise session twice per week. Training program included both aerobic and muscle strength exercises. The 3-month intervention was followed by 3-month follow-up of self-administered exercise. Control group received the same introduction to exercise and training instruction as the supervised group but exercised independently for the entire study period. Fasting cholesterol, plasma glucose, and body composition were evaluated at the baseline, 3 months after randomization, and at the end of the study, 6 months after randomization. Physical activity during the study period was measured using activity bracelets (Polar A370). Study protocol was approved by Pirkanmaa hospital ethics committee (R18021) and registered to Clinicaltrials.gov (NCT04050397). Results: Median total cholesterol increased during 6-month follow-up in both groups, but less in the supervised group compared to the control group (0.1 vs 0.4 mmol/l, respectively, p=0.02). LDL remained stable in supervised group but increased slightly in the control group (0 vs. 0.4 mmol/l, respectively, p=0.01). Fasting plasma glucose lowered slightly in both groups (-0.2 vs -0.4 mmol/l). Total active time per day was slightly higher in the supervised group compared to the control group after 3-month intervention (median 364 vs 293 min/day, respectively). This difference persisted after intervention stopped (median 331 vs 267 min/day, respectively). Median change in body fat percentage at 6 months was 0 in the supervised arm and -0.9 in the control arm. None of the participants dropped out due to exercise-related side-effects. Conclusion: This pilot study showed that it is safe to perform strength exercise in men with ADT. Supervised exercise is likely more effective in mitigating metabolic adverse effect of ADT and inducing long-term changes in physical activity than self-administered exercise. Larger studies with longer follow-up are needed to investigate whether this leads to survival advantage. Citation Format: Lauri Rantaniemi, Aino Siltari, Eeva Harju, Teemu J. Murtola. Can supervised exercise impact on metabolic markers and physical activity during androgen-deprivation therapy in prostate cancer patients? - randomized controlled pilot trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT171.

Introduction: Solid tumors typically favor non-oxygen related pathways on utilizing energy for cell metabolism. This helps tumor cells to survive in hypoxic microenvironment which is common in solid tumors such as prostate cancer (PCa). PCa cells characteristically store lipids, especially cholesterol. Furthermore, hypoxia and lipid metabolism are both associated with decreased responses to immune system and resistance to cancer treatments. Aim of this study was to investigate the impact of hypoxia on cell proliferation and lipid metabolism in advanced PCa cells at various stages of treatment resistance. Material and Methods: PCa cells, VCaPs, were cultured for months to form several unique, treatment resistant advanced PCa cell lines as follows: first, cells were cultured with high (10 nM) testosterone level until they required testosterone for growth to form testosterone sensitive cell line (VCaP-T, demonstrating metastatic hormone sensitive PCa); second, VCaP-T cells were cultured further under low testosterone (0.1 nM) until they gained ability to grow at low androgen level, forming castration resistant cell line (VCaP-CT); third, VCaP-CT cells were further cultured with bicalutamide and/or enzalutamide, androgen signaling inhibitors, until they again gained ability to grow despite the treatment, forming cell lines resistant to these drugs (VCaP-CT-BR, VCap-CT-ER, and VCaP-CT-BR-ER, demonstrating bicalutamide-, enzalutamide-, or mutually resistant cell lines, respectively).All cell lines were exposed to hypoxia (3% O2) with or without simvastatin (1 and 2.5 μM) for 3 days and compared to the cells growing in normoxia. Changes in proliferation was measured using crystal violet staining and amount of intracellular lipids were evaluated using Oil Red O staining. Results: Hypoxia decreased cell proliferation compared to normoxia by 20-25% in VCaP-T, VCaP-CT, and VCaP-CT-BR cells, but had no impact in enzalutamide-resistant cells VCaP-CT-ER or VCaP-CT-BR-ER. Simvastatin treatment decreased cell proliferation in all cell lines equally in hypoxia and normoxia.Compared to androgen-sensitive VCaP-T cells, all treatment resistant cell lines displayed greater intracellular lipid accumulation. Further analyses will address whether hypoxia and/or simvastatin treatment impacts amount of intracellular lipids and whether inhibition of lipid accumulation leads to less tolerance to hypoxia. Conclusion: Enzalutamide resistant cell lines displayed greater hypoxia tolerance compared to androgen sensitive or treatment naïve castration resistant cells. This indicates these cells are adopted to hypoxic microenvironment. All treatment resistant cell lines had greater intracellular lipid accumulation compared to testosterone-sensitive cells, suggesting importance of lipid metabolism in treatment resistance and hypoxia tolerance. Citation Format: Aino Siltari, Merja Bläuer, Heimo Syvälä, Teuvo L. Tammela, Teemu J. Murtola. Differential resistance to hypoxia is linked to increased lipid accumulation in treatment resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 121.

Objectives: To determine whether macroscopic haematuria predicts urethrovesical anastomotic leakage after robot-assisted laparoscopic radical prostatectomy (RALP) as well as a cystogram.Patients an...

The etiology of prostate cancer (PCa) involves environmental and genetic factors. Understanding the role of medication use on PCa risk may clarify the pathophysiological changes and mechanisms in development of cancer.This study investigated PCa risk in relation to overall use of anti-hypertensive drugs and those with specific mechanisms of action. The study cohort (78,615 men) was linked to the prescription database to obtain information on medication use during 20-year follow-up. Information was obtained on PCa diagnoses, causes of deaths, and for a sub-set on B.M.I. and use of non-prescription drugs. Time-dependent drug use variables hazard ratios (HR) with 95% confidence intervals (CI) were calculated using Cox regression analyses.Use of antihypertensive drugs slightly increased PCa risk (HR = 1.16, 95% CI = 1.11-1.22). The risk increase was clearest for metastatic PCa (HR = 1.36, 95% CI = 1.14-1.62). ACE inhibitors, beta-blockers, and diuretics were all separately associated with a small excess risk (HR = 1.10, 95% CI = 1.01-1.19, HR = 1.14, 95% CI = 1.06-1.21, and HR = 1.16, 95% CI = 1.07-1.27, respectively). None of the other groups showed a clear association with PCa risk.The use of antihypertensive drugs was associated with increased prostate cancer risk. Similar risk association for multiple drug groups suggests that the findings may not reflect a direct medication effect, but may be due to underlying hypertension.

The renin angiotensin system (RAS) participates in inflammatory processes, as either a pro-inflammatory or an anti-inflammatory mediator. Components of RAS, such as angiotensin-converting enzyme (ACE), have been detected locally in the gut epithelium. In addition, the anti-inflammatory steroid, corticosterone, is produced in the gut. Hypertension and aging evoke a low-grade inflammatory process in the vascular endothelium. It is not known whether they induce a similar low-grade inflammation in the intestine and if the low-grade inflammation would evoke an activation of ACE and an elevation of corticosterone production. These two variables were measured in ileum and colon of 9- and 26-week old spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WK) controls. ACE-activity, measured via the formation of histidyl-leucine from hippuryl-histidyl-leucine in the tissue homogenate samples, was similar in the ileum and colon of young animals although the ileum of the young normotensive animals displayed the lowest level. In the old animals, the ACE activity was higher in the ileum than in the colon, especially in normotensive rats. Corticosterone production was measured as corticosterone concentration in the supernatants of ileum or colon after a 90-min ex vivo incubation. Corticosterone production was higher in ileum than in colon in both SHR and WK. No clear evidence was seen for age-dependency or for an effect of hypertension in the measured variables in the intestine. Thus, the putative low-grade inflammation in the intestine in aging or hypertension is not a strong enough stimulus to elevate corticosterone production or activate ACE.

Atherosclerosis is a pathological process underpinning many cardiovascular diseases; it is the main cause of global mortality. Atherosclerosis is characterized by an invasion of inflammatory cells, accumulation of lipids and the formation of fatty streaks (plaques) which subsequently allow accumulation of calcium and other minerals leading to a disturbance in the vascular endothelium and its regulatory role in arterial function. Vascular calcification is a different process, stringently regulated mainly by local factors, in which osteoblast-like cells accumulate in the muscular layer of arteries ultimately taking on the physiological appearance of bone. The elevated stiffness of the arteries leads to severe vascular complications in brain, heart and kidneys. Recently, evidence from animal experiments as well as clinical and epidemiological results suggests that long-term treatment with warfarin, but not with the novel direct anticoagulants, can increase the risk or even induce vascular calcification in some individuals. Gamma-carboxylation is an enzymatic process not only needed for activation of vitamin K but also other proteins which participate in bone formation and vascular calcification. Thus, reduced expression of the vitamin K-dependent proteins which physiologically inhibit calcification of cellular matrix could be postulated to lead to vascular calcification. Published clinical data, describing at present a few thousand patients, need to be supplemented with controlled studies to confirm this interesting hypothesis.

In the present study, we aimed to evaluate the effect of aging on the absorption of small peptides in spontaneously hypertensive rats (SHRs). Three kinds of dipeptides, glycyl-sarcosine (Gly-Sar), Trp-His, and captopril (a dipeptidomimetic drug), a Gly-Sar-Sar tripeptide, a Gly-Sar-Sar-Sar tetrapeptide, and a Gly-Sar-Sar-Sar-Sar pentapeptide were administered at doses of 10 mg/kg each to 8- and 40-week-old SHRs. The peptides were all detected in their intact forms in the blood. There was a significantly promoted absorption of di/tripeptides in aged SHRs compared with young SHRs. In contrast, the absorption of tetra/pentapeptides was not affected by aging. PepT1 expression in the mid-jejunum was significantly increased in 40-week-old SHRs compared with 8-week-old SHRs, whereas aging did not alter the expression of claudin-1, a tight junction related protein. Thus, the present results suggest that SHR aging may enhance the absorption of di/tripeptides through the enhanced PepT1 transport route, although oligopeptides may be absorbed in an age-independent manner.

Introduction Erectile dysfunction is common after radical prostatectomy because of damage to the cavernous nerves. Thus, it is important to identify new ways to avoid this problem. For example, statins have shown positive effects on erectile function and may have anti-inflammatory effects that improve recovery after surgery. Aim The aim of this exploratory analysis of a subgroup from ESTO1, a randomized, double-blind, placebo-controlled study, was to evaluate the preoperative use of atorvastatin on erectile function after radical prostatectomy. Method Patients were randomized to either 80 mg atorvastatin or placebo daily before undergoing radical prostatectomy from study inclusion to the day of surgery. Altogether 118 men with prostate cancer and scheduled for radical prostatectomy were asked to fill out the 5-item version of the International Index of Erectile Function (IIEF-5) questionnaire before surgery and at 3, 6, 9, and 12 months after surgery. Main Outcome Measurements The study was exploratory, with the main outcome being the overall difference between IIEF-5 scores in the 2 groups at 12 months. Several hypotheses generating sub-analyses were conducted. Results Overall, 85% filled out the IIEF-5 questionnaire before their operation and 85%, 81%, 78%, and 78% completed it at 3, 6, 9, and 12 months follow-up, respectively. 52% of men had information available at all time points. There were no statistically significant differences between the groups at baseline in either erectile function, comorbidities, or tumor characteristics. The median duration of use of atorvastatin and placebo before surgery was 27 and 25 days, respectively. Preoperative atorvastatin treatment had no statistically significant effect on erectile function after prostatectomy as compared with placebo, although IIEF-5 scores were higher at all time points in the statin arm. Furthermore, atorvastatin treatment compared with placebo improved IIEF-5 scores at 12 months after surgery when the cavernous nerves were at least partially intact bilaterally (P < .04, n = 65); however, after full bilateral or unilateral nerve-sparing, the difference was not statistically significant. Clinical Implication Short-term statin treatment did not improve recovery of erectile function after prostatectomy; however, further studies are needed before final conclusions. Strengths & Limitations This was a randomized placebo-controlled study. Original ESTO1 study was designed to detect a difference in prostate cancer biomarkers. Conclusion Short-term atorvastatin treatment before radical prostatectomy had no statistically significant effect on the recovery of erectile functions in a non-selected cohort of patients undergoing radical prostatectomy. Further studies will be needed to clarify the role of long-term atorvastatin use before and after prostatectomy.

Simple Summary Pharmacoepidemiologic research provides opportunities to evaluate how commonly used drug groups, such as cholesterol-lowering drugs, may affect the prostate cancer risk or mortality. However, such studies need to be carefully designed in order to avoid biases caused by systematic differences between medication users and non-users. Similarly, data must be carefully analyzed and interpreted while acknowledging possible biases that can lead to erroneous conclusions. Here, we review common pitfalls in such studies and describe ways to avoid them in an effort to aid future research. Abstract Pharmacoepidemiologic research provides opportunities to evaluate how commonly used drug groups, such as cholesterol-lowering or antidiabetic drugs, may affect the prostate cancer risk or mortality. This type of research is valuable in estimating real-life drug effects. Nonetheless, pharmacoepidemiological studies are prone to multiple sources of bias that mainly arise from systematic differences between medication users and non-users. If these are not appreciated and properly controlled for, there is a risk of obtaining biased results and reaching erroneous conclusions. Therefore, in order to improve the quality of future research, we describe common biases in pharmacoepidemiological studies, particularly in the context of prostate cancer research. We also list common ways to mitigate these biases and to estimate causality between medication use and cancer outcomes.

High salt in diet elevates blood pressure in man. Berries and fruits are regarded beneficial in antagonizing hypertension and related vascular complications. We aimed to test whether lingonberry juice shows beneficial and anti-inflammatory effects in salt induced hypertension rat model. Male Wistar–Kyoto rats were fed with 8% sodium chloride enriched pellets for 10 weeks having tap water or diluted lingonberry juice as drinking fluid. Blood pressure was recorded weekly and mesenteric artery functions tested ex vivo. Urine was collected in metabolic caging. Salt loading had only minor effects on blood pressure or endothelial function. Increased urine excretion of 8-isoprostane, cGMP and albumin reflected general or renal inflammation. Elevated expression (mRNA) of proinflammatory COX-2 by salt was normalized by lingonberry. Salt increased kidney, heart and left ventricle masses and changed serum chloride, alkaline phosphatase, albumin and lipid concentrations. Taken together the results show that l0 weeks’ high-salt diet impaired kidney function of young rats without clear effect on blood pressure or vascular function. Lingonberry juice moderately reduced biomarkers of low-grade inflammation probably due to its high polyphenol concentrations.

Given that vascular calcification is inversely correlated with the clinical intake of menaquinone, a rat model of warfarin-induced calcification may be useful for testing menaquinone and vitamin K-1 potential effects on vascular function. The aim of the present study was to investigate effects of vitamin K-1 and menaquinone-7 treatments on blood pressure and vascular function in warfarin-induced vascular calcification model during five-week intervention in normotensive Wistar-Kyoto rats. Blood pressure was measured weekly, and at the end of the intervention in vitro vascular reactivity measurements were done. Alizarin Red S and von Kossa stainings were used to record possible calcification of aortic sections. Routine clinical chemistry was done from serum and urine samples. Vascular calcification was seen only in a few warfarin-treated animals in histological staining. Warfarin-treatment did not change significantly blood pressure of the rats. Warfarin-treatment increased slightly the endothelium-dependent relaxation of aorta after the L-type calcium channels were blocked. Also the vascular relaxation improved after NOS inhibition in the aorta of the healthy controls and menaquinone-7 treated animals, indicating that the relaxation in those groups was not totally dependent on NO. Clinical chemistry from serum showed some differences in urea, creatinine as well as lipid and glucose metabolism between the healthy controls and warfarin-treated rats.

Lingonberries ( Vaccinium vitis-idaea ) are rich in polyphenols, such as proanthocyanidins, anthocyanins, flavonols and flavanols. Polyphenol-rich foods affect vascular health. We previously described improved endothelium-dependent vascular function as well as anti-inflammatory and anti-atherothrombotic effects in spontaneously hypertensive rats (SHR) fed with lingonberry juice. In the present study, we investigated the effects of lingonberry juice dose on blood pressure, vascular function and vascular inflammation in SHR in an 8 weeks’ study. Diluted (1:5) cold-compressed lingonberry juice was given as drinking fluid ad libitum to 5 week old SHR for 8 weeks. Control group (SHR) and normotensive reference group (Wistar-Kyoto) received tap water. Systolic blood pressure (SBP) was monitored weekly. Function of mesenteric artery rings was assessed in standard organ-bath chambers after 8 weeks. Biochemical and clinical chemistry variables were measured from plasma and urine. Lingonberry juice lowered SBP of SHR ( p = 0.007). Endothelium-dependent vascular relaxation was not improved. Lingonberry treatment slightly affected plasma inflammatory markers (reduction of NOx and sICAM-1) and clinical chemistry variables (decreased alkaline phosphatase and increased chloride and calcium levels). In conclusion, Lingonberry juice at small concentrations lowers blood pressure in a long-term treatment in SHR.

Bradykinin exerts its vascular actions via two types of receptors, the non-constitutively expressed bradykinin receptor type 1 (BR1) and the constitutive type 2 receptor (BR2). Bradykinin-induced vasorelaxation is age-dependent, a phenomenon related to the varying amounts of BR1 and BR2 in the vasculature. Isoleucine-proline-proline (Ile-Pro-Pro), a bioactive tripeptide, lowers elevated blood pressure and improves impaired endothelium-dependent vasorelaxation in hypertensive rats. It inhibits angiotensin converting enzyme 1 (ACE1). Other mechanisms of action have also been postulated. The aims of the study were to clarify the underlying mechanisms of the age-dependency of bradykinin-induced vasodilatation such as the roles of the two bradykinin receptors, themas-receptor and synergism with Ile-Pro-Pro. The vascular response studies were conducted using mesenteric artery and aorta rings from normotensive 6 wk. (young) and 22 wk. (old) Wistar rats. Cumulative dosing of acetylcholine, bradykinin and angiotensin(1-7) (Ang(1-7))were tested in phenylephrine-induced vasoconstriction with or without 10 min pre-incubation with antagonists against BR1-, BR2- or mas-receptors,Ang(1-7) or ACE1-inhibitors captopril and Ile-Pro-Pro. The bradykinin-induced vasorelaxation in vitro was age-dependent and it was improved by pre incubation with Ile-Pro-Pro, especially in old rats with endothelial dysfunction. The mas-receptor antagonist, D-Pro7-Ang(1-7) abolished bradykinin-induced relaxation totally. Interestingly, BR1 and BR2 antagonists only slightly reduced bradykinin-induced vasorelaxation, as an evidence for the involvement of other mechanisms in addition to receptor activation. In conclusion, bradykinin-induced vasorelaxation was age -dependent and He-Pro-Pro improved it. Mas receptor antagonist abolished relaxation while bradykinin receptor antagonist only slightly reduced it, suggesting that bradykinin-induced vasorelaxation is regulated also by other mechanisms than the classical BR1/BR2 pathway. (C) 2016 Elsevier Inc: All rights reserved.

Flavonoids and other phenolic compounds affect low-grade inflammation related to cardiovascular diseases among other positive health effects. Cardioprotective actions are mainly due to enhanced endothelial function and production of nitric oxide (NO). We investigated vascular anti-inflammatory effects of cranberry (Vaccinium oxycoccos), lingonberry (Vaccinium vitis-idaea) and blackcurrant (Ribes nigrum) juices given as drinking fluid ad libitum to spontaneously hypertensive rats (SHR), a widely used model of human hypertension, in an 8 week intervention study. The animals were sacrificed, the aortas cleaned and RNA was extracted. cDNA was prepared for real-time PCR and blood was collected for biochemical analyses. The mRNA expressions of angiotensin-converting enzyme 1 (ACE1), cyclooxygenase 2 (COX2), monocyte chemoattractant protein 1 (MCP1) and P-selectin were significantly reduced in the cranberry and lingonberry groups. These findings suggest that cranberry and lingonberry cold-compressed juices have anti-inflammatory and anti-atherothrombotic actions in long-term treatment of SHR.

In this study, we investigated the synergistic effects of plant sterols (PS) and casein-derived tripeptides on arterial tone and blood pressure in experimental hypertension. We hypothesized that PS and tripeptides could have positive, synergistic effects on the development of hypertension and endothelial dysfunction in young spontaneously hypertensive rats (SHR). Six-week-old male SHR were divided into 3 groups to receive milk products containing PS, or PS with tripeptides, or a control containing no active components for 8 weeks. Systolic blood pressure (SBP) was measured weekly, and vascular reactivity measurements with isolated mesenteric arteries were performed at the end of the study. Biochemical measurements for several parameters were performed by enzyme-linked immunosorbent assay using plasma samples. Levels of angiotensin-converting enzyme 1, cyclooxygenase-2, endothelial nitric oxide synthase, and P-selectin messenger RNA expressions were determined from aortic tissue by real-time polymerase chain reaction. The study showed that long-term treatment with PS + tripeptides attenuated the development of hypertension in SHR (SBP, 187 ± 5 mm Hg vs 169 ± 4 mm Hg in control group; P < .01). Plant sterols alone did not affect SBP significantly. Endothelial dysfunction was observed in all SHR; however, treatment with PS resulted in poorer endothelium-dependent and nitric oxide–mediated relaxation compared with other groups. Aortic cyclooxygenase-2 and P-selectin were significantly down-regulated in PS and PS + tripeptides groups when compared with the control group. The expression of endothelial nitric oxide synthase was significantly lower in PS than in PS + tripeptides group. In conclusion, long-term treatment with PS has a slight but not significant antihypertensive effect. Plant sterols do not provide any beneficial effects on endothelial function in hypertensive rats; however, treatment with both PS and tripeptides showed mild anti-inflammatory effects.

In the fermentation of milk by certain lactic acid bacteria, casein is degraded into bioactive tripeptides shown to lower blood pressure in experimental animal models and in mildly hypertensive humans. This effect is suggested to result mainly in inhibition of angiotensin converting enzyme 1 (ACE-1). Due to the complexity of renin-angiotensin system (RAS), several other enzymes than ACE-1 can participate in the production of vasoactive components. Therefore, in the present study we investigated effects of tripeptides isoleucine-proline-proline (IPP), valine-proline-proline (VPP) and leucine-proline-proline (LPP) on some endothelial enzymes that are important in RAS or otherwise have a role in the endothelial function. The enzymes investigated were renin, chymase, neutral endopeptidase (NEP), prolyl oligopeptidase (POP), cathepsin G, endothelin converting enzyme 1 (ECE-1), and cyclooxygenase 1 and 2 (COX -1 and COX-2). The tripeptides inhibited prolyl oligopeptidase (POP) dose-dependently. IPP was the most potent inhibitor (IC 50 486±95 µM). Contrary, cathepsin G was activated by IPP, VPP and LPP as well as the amino acids proline and isoleucine. The other investigated enzymes were not affected. Inhibition of POP and activation of cathepsin G do not explain the blood pressure lowering effects of the tripeptides. Thus the inhibition of ACE-1 remains the most plausible mechanism of the antihypertensive effects of the tripeptides.

Aino Siltari,1 Riikka Viitanen,2 Sampo Kukkurainen,2 Heikki Vapaatalo,1 Jarkko Valjakka2 1Institute of Biomedicine, Pharmacology, University of Helsinki, Finland; 2BioMediTech, Institute of Biomedical Technology, University of Tampere, Finland Background: The milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature. Purpose: We focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition. Methods: AutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures. Results: In HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings. Keywords: ACE inhibition, Autodock modeling, Ile-Pro-Pro, Val-Pro-Pro, vascular function

Cardiovascular diseases and hypertension are common in the aging population. Nutritional life-style factors can help to reduce and maintain a healthy level of blood pressure. Two milk-derived tripeptides, Ile-Pro-Pro and Val-Pro-Pro, have been shown to decrease elevated blood pressure in humans and in experimental models. They also improve endothelial dysfunction and thus promote vascular health. Although their principal blood pressure lowering mechanism is thought to be inhibition of the angiotensin-converting enzyme (ACE), other mechanisms are also possible. ACE is one of the key enzymes in the renin-angiotensin system (RAS) which is linked with bradykinin metabolism. In this thesis, the two tripeptides’ antihypertensive mechanisms were examined using an experimental model of hypertension, spontaneously hypertensive rats (SHR). There was a special focus on bradykinin and its role in vascular reactivity in hypertensive and ageing animals. Furthermore, we evaluated whether the tripeptides possess other mechanism(s) of action in addition to ACE inhibition. We also applied computerized modelling to elucidate the possible role of different cis/trans stereoisomers of the tripeptides in ACE inhibition. Finally, we investigated transport of the tripeptides across cell membrane by modelling this process in caco-2 cells. Long term feeding of the tripeptides retarded the development of hypertension during aging of both SHR and healthy old animals. Bradykinin induced vasorelaxation in young healthy normotensive animals; this was abolished in old animals. However, pre-incubation in vitro with Ile-Pro-Pro before exposure to bradykinin exerted a synergistic effect with bradykinin to induce vasorelaxation even in old animals. Low-grade inflammation in the vasculature has been shown to occur during ageing as well as being present in hypertension. We suggest that this converts the normal bradykinin property to relax blood vessels into a vasoconstrictive effect. Our results show that this phenomenon is related to the production of vasoconstrictive prostanoids in the endothelium. Tripeptides are able to inhibit and activate also other enzymes than ACE at least in in vitro. Our computerized model revealed that cis-trans is the best stereoisomer configuration of the tripeptide to inhibit ACE1. Tripeptides penetrated across the caco-2 cell monolayer in an intact form using both paracellular and peptide transporter 1 (Pept1) mediated routes. The results of the thesis strongly support the concept that the bioactive tripeptides and probably other small molecular peptides from different dietary components such as milk and fish, should be investigated in long-term controlled clinical trials in an elderly population instead of the previously examined young or middle-aged individuals. Special focus should be placed on vascular function which has been a poorly investigated area and has been demonstrated to be improved by the tripeptides, especially in old animals. Joka vuosi sydän- ja verisuonisairaudet aiheuttavat miljoonien ihmisten ennenaikaisen kuoleman Euroopassa. Korkea verenpaine on yksi päätekijä, joka hoitamattomana johtaa sydän- ja verisuonisairauksien syntyyn. Huolimatta olemassa olevista lääkkeistä verenpainetaudin hoitoon tarvitaan uusia keinoja, joilla ei olisi lääkkeiden haittoja. Yksi varteenotettava vaihtoehto tai lisä lääkehoidon ohella voisi olla ravinnosta saatavat luontaiset tekijät. Maito on korkean proteiinipitoisuutensa ansiosta erinomainen lähde proteiineista pilkkoutuville lyhyille aminohappoketjuille eli peptideille. Tällaisia bioaktiivisiksiksi peptideiksi kutsuttuja yhdisteitä voi pilkkoutua maidon proteiineista mm. maitohappobakteerien vaikutuksesta. Tällaisia peptideitä ovat esimerkiksi kolmen aminohapon mittaiset tripeptidit, isoleusiini-proliini-proliini (Ile-Pro-Pro) ja valiini-proliini-proliini (Val-Pro-Pro). Niiden on osoitettu alentavan kohonnutta verenpainetta kokeellisissa malleissa ja kliinisissä tutkimuksissa lievästi korkea verenpaineisilla henkilöillä. Ensisijaiseksi vaikutusmekanismiksi on osoitettu, lääkekohteenakin toimivan, angiotensiiniä konvertoivan entsyymin (ACE) aktiivisuuden vähentäminen ja sitä kautta voimakkaasti verisuonia supistavan angiotensiini II:n muodostumisen esto. Mainituilla pienillä peptideillä uskotaan olevan myös muita vaikutusmekanismeja sydämen ja verisuoniston toimintaan osallistuviin tekijöihin. Helsingin yliopiston lääketieteellisen tiedekunnan lääketieteellisen ravitsemusfysiologian ryhmä on useiden vuosien ajan tehnyt verenpaineeseen ja verisuonten toimintaan kohdistuvaa tutkimusta. Tämä työ on jatkoa sille ja sen tarkoituksena oli selvittää tripeptidien verenpaineeseen ja verisuoniston toimintaan liittyviä toimintamekanismeja erilaisissa kokeellisissa malleissa. Työssä keskityttiin iän ja verenpaineen kohoamiseen liittyvään verisuonten lievään tulehdukseen sekä solumalleihin. Tutkimuksessa keskityttiin verisuonien jäykkyyttä sääteleviin fysiologisiin tekijöihin ja niiden kohdemolekyyleihin. Tutkimuksessa käytettiin myös tietokonemallinnusta selvittämään Ile-Pro-Pro:n aminohappojen välisten sidosten (cis/trans) avaruudellisen rakenteen merkitystä kohdennettaessa yhdisteen sitoutumista ACE:n aktiiviseen keskukseen. Voidakseen päästä vaikutuspaikkaansa verisuonen seinämään peptidien on imeydyttävä suolistosta, joten imeytymistä selvitettiin käyttäen suolen pintasolukkoa mallintavia soluviljelmiä. Päätulokset osoittivat, että tripeptidit alensivat korkeaverenpaineisen eläimen verenpainetta juomaan lisättynä pitkäaikaisessa kokeessa. Niiden todettiin vaikuttavan myös ACE-eston lisäksi joihinkin muihin verisuonten entsyymeihin ja reseptoreihin, mikä voi osaltaan selittää tripeptideillä kliinisestikin havaittua verenpaineen alenemista ja verisuonten jäykkyyttä kuvaavien mittareiden paranemista. Tietokonemallinnus osoitti, että tutkitun peptidin neljästä avaruudellisesta muodosta cis-trans muoto on edullisin ACE-estossa. Tripeptidit imeytyvät suoliston solukerroksen läpi pilkkoutumatta käyttäen hyväkseen sekä aktiivista että passiivista kuljetusta. Tutkimuksen tulokset osoittavat, että ravinnon komponenteilla, tässä tapauksessa maidon proteiineista pilkkoutuvilla peptideillä, saattaa pitkäaikaisessa käytössä olla merkitystä sydämen ja verisuoniston terveydelle. Aikaisempien kliinisten tutkimusten ja tämän väitöskirjatyön perusteella niistä voisi olla hyötyä, kun suositellaan verenpainetta seurattaessa potilaalle ravitsemuksellisia valintoja.