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Background: Low-intensity pulsed ultrasound (LIPUS), as a safe biophysiotherapy, can enhance bone-tendon (B-T) healing in vivo and induce osteogenic or chondrogenic differentiation of mesenchymal stromal cells in vitro. This study aimed to determine whether LIPUS can improve the efficacy of transplanted mesenchymal stromal cells on B-T healing. Hypothesis: LIPUS can induce lineage-specific differentiation of transplanted adipose-derived stromal cells (ASCs) at the B-T healing site, thus resulting in superior healing quality when compared with LIPUS or ASCs alone. Study Design: Controlled laboratory study. Methods: A total of 112 mature rabbits with partial patellectomy in the hindlimb were randomly assigned into mock sonication without ASCs (control), ultrasonication without ASCs (LIPUS), mock sonication with ASCs (ASCs), and ultrasonication with ASCs (LIPUS + ASCs). The treatment time of the mock sonication or ultrasonication was 20 minutes per day. Autologous ASCs were transplanted to the healing site by fibrin glue during the operation, and LIPUS was delivered daily starting at postoperative day 3 until euthanasia. The patella–patellar tendon junctions were postoperatively harvested at 8 and 16 weeks for radiological, histological, and mechanical evaluations. Additionally, 9 animals were used for ASC tracking with mCherry protein. Results: Radiologically, there was more new bone formation and remodeling in the LIPUS + ASCs group as compared with the other groups. Synchrotron radiation micro–computed tomography showed that the LIPUS + ASCs group significantly increased bone volume fraction, trabecular thickness, and trabecular number at the healing site as compared with the other groups at postoperative 8 weeks ( P < .05 for all). Histologically, immunohistochemical staining confirmed that the transplanted mCherry-ASCs can differentiate into osteoblasts and fibrochondrocytic-like cells. Meanwhile, as compared with the other groups, the LIPUS + ASCs group showed more formation and maturity of the fibrocartilage layer and new bone at postoperative weeks 8 and 16 ( P < .05 for all). Biomechanically, the LIPUS + ASCs group showed significantly higher failure load and stiffness versus the other groups at postoperative weeks 8 and 16 ( P < .05 for all). Conclusion: Autologous ASC transplantation stimulated with LIPUS can result in superior B-T healing quality when compared with LIPUS or ASCs alone. Clinical Relevance: This study demonstrates the effectiveness of using ASC transplantation stimulated with LIPUS for B-T healing and provides a foundation for future clinical studies.

Despite optimal current therapies, cardiovascular disease remains the leading cause for death worldwide. Importantly, advances in peptide engineering have accelerated the development of innovative therapeutics for diverse human disease states. Additionally, the advancement of bispecific therapeutics targeting >1 signaling pathway represents a highly innovative strategy for the treatment of cardiovascular disease. We, therefore, engineered a novel, designer peptide, which simultaneously targets the pGC-A (particulate guanylyl cyclase A) receptor and the MasR (Mas receptor), potentially representing an attractive cardiorenoprotective therapeutic for cardiovascular disease. We engineered a novel, bispecific receptor activator, NPA7, that represents the fusion of a 22-amino acid sequence of BNP (B-type natriuretic peptide; an endogenous ligand of pGC-A) with Ang 1–7 (angiotensin 1–7)—the 7-amino acid endogenous activator of MasR. We assessed NPA7’s dual receptor activating actions in vitro (second messenger production and receptor interaction). Further, we performed an intravenous peptide infusion comparison study in normal canines to study its biological actions in vivo, including in the presence of an MasR antagonist. Our in vivo and in vitro studies demonstrate the successful synthesis of NPA7 as a bispecific receptor activator targeting pGC-A and MasR. In normal canines, NPA7 possesses enhanced natriuretic, diuretic, systemic, and renal vasorelaxing and cardiac unloading properties. Importantly, NPA7’s actions are superior to that of the individual native pGC-A or MasR ligands. These studies advance NPA7 as a novel, bispecific designer peptide with potential cardiorenal therapeutic benefit for the treatment of cardiovascular disease, such as hypertension and heart failure.

Background: To describe the baseline characteristics of patients with heart failure and preserved left ventricular ejection fraction enrolled in the PARAGON-HF trial (Prospective Comparison of Angiotensin Receptor Neprilysin Inhibitor With Angiotensin Receptor Blocker Global Outcomes in HFpEF) comparing sacubitril/valsartan to valsartan in reducing morbidity and mortality. Methods and Results: We report key demographic, clinical, and laboratory findings, and baseline therapies, of 4822 patients randomized in PARAGON-HF, grouped by factors that influence criteria for study inclusion. We further compared baseline characteristics of patients enrolled in PARAGON-HF with those patients enrolled in other recent trials of heart failure with preserved ejection fraction (HFpEF). Among patients enrolled from various regions (16% Asia-Pacific, 37% Central Europe, 7% Latin America, 12% North America, 28% Western Europe), the mean age of patients enrolled in PARAGON-HF was 72.7±8.4 years, 52% of patients were female, and mean left ventricular ejection fraction was 57.5%, similar to other trials of HFpEF. Most patients were in New York Heart Association class II, and 38% had ≥1 hospitalizations for heart failure within the previous 9 months. Diabetes mellitus (43%) and chronic kidney disease (47%) were more prevalent than in previous trials of HFpEF. Many patients were prescribed angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (85%), β-blockers (80%), calcium channel blockers (36%), and mineralocorticoid receptor antagonists (24%). As specified in the protocol, virtually all patients were on diuretics, had elevated plasma concentrations of N-terminal pro-B-type natriuretic peptide (median, 911 pg/mL; interquartile range, 464–1610), and structural heart disease. Conclusions: PARAGON-HF represents a contemporary group of patients with HFpEF with similar age and sex distribution compared with prior HFpEF trials but higher prevalence of comorbidities. These findings provide insights into the impact of inclusion criteria on, and regional variation in, HFpEF patient characteristics. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01920711.

The particulate guanylyl cyclase A (pGC-A)/cGMP pathway plays important roles in regulating renal physiological function and as well as in counteracting pathophysiological conditions. Naturally occurring peptide pGC-A activators consist of atrial natriuretic peptide (ANP), b-type NP (BNP), and urodilatin (URO). These activators bind and activate pGC-A, generating the second messenger cyclic 3′,5′ guanosine monophosphate (cGMP). Cyclic GMP binds to downstream pathway effector molecules including protein kinase G (PKG), cGMP-gated ion channels, and phosphodiesterases (PDEs). These mediators result in a variety of physiological actions in the kidney, including diuresis, natriuresis, increased glomerular filtration rate (GFR) and organ protection, thus, opposing renal cellular injury and remodeling. Downstream proteins regulated by PKG include collagen 1 (Col-1), transforming growth factor beta (TGF-β) and apoptosis-related proteins. In addition to their physiological regulatory effects, pGC-A/cGMP signaling is critical for preserving renal homeostasis in different renal diseases such as acute kidney injury (AKI). Regarding therapeutic options, native pGC-A activators have short half-lives and their activity can be further enhanced by advances in innovative peptide engineering. Thus, novel designer peptide pGC-A activators with enhanced renal activity are under development.

The natriuretic peptides (NPs) B-type NP (BNP) and urodilatin (URO) exert renal protective properties via the particulate guanylyl cyclase A receptor (pGC-A). As a potential renal-enhancing strategy, we engineered a novel designer peptide that we call CRRL269. CRRL269 was investigated in human cell lines and in normal canines to define potential cardiorenal enhancing actions. The mechanism of its cardiorenal selective properties was also investigated. In vitro NP receptor activity was quantified with guanosine 3′,5′-cyclic monophosphate generation. In vivo effects were determined in normal canine acute infusion studies. We observed that CRRL269 demonstrated enhanced pGC-A activity in renal compared with nonrenal cell lines. CRRL269 exerted enhanced resistance to neprilysin compared with URO. Importantly, CRRL269 exhibited significant and greater increases in urinary sodium excretion and diuresis, with less blood pressure reduction, than BNP or URO in normal canines. CRRL269 retained potent renin-angiotensin-aldosterone system (RAAS) suppressing properties shared by URO and BNP. Also, CRRL269 exerted less arterial relaxation and higher cAMP cardiomyocytes generation than BNP. CRRL269 possessed superior renal and pGC-A activating properties compared with BNP or URO in vitro. CRRL269 exerted enhanced renal actions while suppressing RAAS in vivo and with less hypotension compared with URO or BNP. Together, our study suggests that CRRL269 is a promising innovative renal-enhancing drug, with favorable protective actions targeting cardiorenal disease states through the pGC-A receptor.

Abstract Breast cancer risks conferred by many germline missense variants in the BRCA1 and BRCA2 genes, often referred to as variants of uncertain significance (VUS), have not been established. In this study, associations between 19 BRCA1 and 33 BRCA2 missense substitution variants and breast cancer risk were investigated through a breast cancer case–control study using genotyping data from 38 studies of predominantly European ancestry (41,890 cases and 41,607 controls) and nine studies of Asian ancestry (6,269 cases and 6,624 controls). The BRCA2 c.9104A&gt;C, p.Tyr3035Ser (OR = 2.52; P = 0.04), and BRCA1 c.5096G&gt;A, p.Arg1699Gln (OR = 4.29; P = 0.009) variant were associated with moderately increased risks of breast cancer among Europeans, whereas BRCA2 c.7522G&gt;A, p.Gly2508Ser (OR = 2.68; P = 0.004), and c.8187G&gt;T, p.Lys2729Asn (OR = 1.4; P = 0.004) were associated with moderate and low risks of breast cancer among Asians. Functional characterization of the BRCA2 variants using four quantitative assays showed reduced BRCA2 activity for p.Tyr3035Ser compared with wild-type. Overall, our results show how BRCA2 missense variants that influence protein function can confer clinically relevant, moderately increased risks of breast cancer, with potential implications for risk management guidelines in women with these specific variants. Cancer Res; 77(11); 2789–99. ©2017 AACR.

Abstract BACKGROUND Neprilysin (NEP) is a membrane-bound neutral endopeptidase that degrades a variety of bioactive peptides. The substrates include natriuretic peptides (NPs), which are important regulating mediators for cardiovascular and renal biology. Inhibition of NEP activity and exogenous NP administration thus have emerged as potential therapeutic strategies for treating cardiorenal diseases. More recently, B-type natriuretic peptide (BNP) or N-terminal-proBNP (NT-proBNP), 3′-5′ cyclic guanosine monophosphate (cGMP), and soluble NEP as biomarkers have also been investigated in heart failure (HF) trials and their predictive value are beginning to be recognized. CONTENT The biological functions of NEP and NPs are discussed. Enhancing NPs through NEP inhibition combined with renin–angiotensin–aldosterone system (RAAS) antagonism has proved to be successful in HF treatment, although future surveillance studies will be required. Direct NP enhancement through peptide delivery may have fewer potentially hazardous effects compared to NEP inhibition. Strategies of combined inhibition on NEP with other cardiorenal pathophysiological pathways are promising. Finally, monitoring BNP/NT-proBNP/cGMP concentrations during NEP inhibition treatment may provide supplemental benefits to conventional biomarkers, and the identification of soluble NEP as a novel biomarker for HF needs further investigation. SUMMARY In this review, the biology of NEP is summarized, with a focus on NP regulation. The degradation of NPs by NEP provides the rationale for NEP inhibition as a strategy for cardiorenal disease treatment. We also describe the current therapeutic strategies of NEP inhibition and NP therapeutics in cardiorenal diseases. Moreover, the discovery of its circulating form, soluble NEP, as a biomarker is also discussed.