Activation of angiotensin converting enzyme 2 promotes hippocampal neurogenesis via activation of Wnt/ß-catenin signaling in hypertension.

Hypertension-induced brain renin-angiotensin system (RAS) activation and neuroinflammation are hallmark neuropathological features of neurodegenerative diseases. Previous studies from our lab have shown that inhibition of ACE/Ang II/AT1R axis (by AT1R blockers or ACE inhibitors) reduced neuroinflammation and accompanied neurodegeneration via up-regulating adult hippocampal neurogenesis. Apart from this conventional axis, another axis of RAS also exists i.e., ACE2/Ang (1-7)/MasR axis, reported as an anti-hypertensive and anti-inflammatory. However, the role of this axis has not been explored in hypertension-induced glial activation and hippocampal neurogenesis in rat models of hypertension. Hence, in the present study, we examined the effect of ACE2 activator, Diminazene aceturate (DIZE) at 2 different doses of 10 mg/kg (non-antihypertensive) and 15 mg/kg (antihypertensive dose) in renovascular hypertensive rats to explore whether their effect on glial activation, neuroinflammation, and neurogenesis is either influenced by blood-pressure. The results of our study revealed that hypertension induced significant glial activation (astrocyte and microglial), neuroinflammation, and impaired hippocampal neurogenesis. However, ACE2 activation by DIZE, even at the low dose prevented these hypertension-induced changes in the brain. Mechanistically, ACE2 activation inhibited Ang II levels, TRAF6-NFκB mediated inflammatory signaling, NOX4-mediated ROS generation, and mitochondrial dysfunction by upregulating ACE2/Ang (1-7)/MasR signaling. Moreover, DIZE-induced activation of the ACE2/Ang (1-7)/MasR axis upregulated Wnt/ß-catenin signaling, promoting hippocampal neurogenesis during the hypertensive state. Therefore, our study demonstrates that ACE2 activation can effectively prevent glial activation and enhance hippocampal neurogenesis in hypertensive conditions, regardless of its blood pressure-lowering effects.

Diminazene aceturate attenuates systemic inflammation via microbiota gut-5-HT brain-spleen sympathetic axis in male mice.

The sympathetic arm of the inflammatory reflex is the efferent pathway through which the central nervous system (CNS) can control peripheral immune responses. Diminazene aceturate (DIZE) is an antiparasitic drug that has been reported to exert protective effects on various experimental models of inflammation. However, the pathways by which DIZE promotes a protective immunomodulatory effects still need to be well established, and no studies demonstrate the capacity of DIZE to modulate a neural reflex to control inflammation. C57BL/6 male mice received intraperitoneal administration of DIZE (2 mg/Kg) followed by lipopolysaccharide (LPS, 5 mg/Kg, i.p.). Endotoxemic animals showed hyperresponsiveness to inflammatory signals, while those treated with DIZE promoted the activation of the inflammatory reflex to attenuate the inflammatory response during endotoxemia. The unilateral cervical vagotomy did not affect the anti-inflammatory effect of DIZE in the spleen and serum. At the same time, splenic denervation attenuated tumor necrosis factor (TNF) synthesis in the spleen and serum. Using broad-spectrum antibiotics for two weeks showed that LPS modulated the microbiota to induce a pro-inflammatory profile in the intestine and reduced the serum concentration of tryptophan and serotonin (5-HT), while DIZE restored serum tryptophan and increased the hypothalamic 5-HT levels. Furthermore, the treatment with 4-Chloro-DL-phenylalanine (pcpa, an inhibitor of 5-HT synthesis) abolished the anti-inflammatory effects of the DIZE in the spleen. Our results indicate that DIZE promotes microbiota modulation to increase central 5-HT levels and activates the efferent sympathetic arm of the inflammatory reflex to control splenic TNF production in endotoxemic mice.

Diminazene aceturate-induced cytotoxicity is associated with the deregulation of cell cycle signaling and downregulation of oncogenes Furin, c-MYC, and FOXM1 in human cervical carcinoma Hela cells.

Diminazene aceturate (DIZE) is an FDA-listed small molecule known for the treatment of African sleeping sickness. In vivo studies showed that DIZE may be beneficial for a range of human ailments. However, there is very limited information on the effects of DIZE on human cancer cells. The current study aimed to investigate the cytotoxic responses of DIZE, using the human carcinoma Hela cell line. WST-1 cell proliferation assay showed that DIZE inhibited the viability of Hela cells in a dose-dependent manner and the observed response was associated with the downregulation of Ki67 and PCNA cell proliferation markers. DIZE-treated cells stained with acridine orange-ethidium and JC-10 dye revealed cell death and loss of mitochondrial membrane potential (Ψm), compared with DMSO (vehicle) control, respectively. Cellular immunofluorescence staining of DIZE-treated cells showed upregulation of caspase 3 activities. DIZE-treated cells showed downregulation of mRNA for G1/S genes CCNA2 and CDC25A, S-phase genes MCM3 and PLK4, and G2/S phase transition/mitosis genes Aurka and PLK1. These effects were associated with decreased mRNA expression of Furin, c-Myc, and FOXM1 oncogenes. These results suggested that DIZE may be considered for its effects on other cancer types. To the best of our knowledge, this is the first study to evaluate the effect of DIZE on human cervical cancer cells.

Pyronaridine combined with diminazene aceturate inhibits Babesia in vitro and in vivo

@#Objective: To evaluate the combination therapy of pyronaridine tetraphosphate and diminazene aceturate against Babesia in vitro and in vivo. Methods: Bioinformatic analysis was performed using atom pair fingerprints. An in vitro combination test was performed against Babesia bovis and Theileria equi. Moreover, the in vivo chemotherapeutic efficacy of pyronaridine tetraphosphate in combination with diminazene aceturate was investigated against the growth of Babesia microti in mice using a fluorescence inhibitory assay. Results: Pyronaridine tetraphosphate and diminazene aceturate exhibited nearly similar molecular weights. The in vitro combination of pyronaridine tetraphosphate and diminazene aceturate was synergistic on Babesia bovis and additive on Theileria equi. In addition, 5 mg/kg pyronaridine tetraphosphate combined with 10 mg/kg diminazene aceturate inhibited Babesia microti growth significantly compared with those observed after treatment with 25 mg/kg diminazene aceturate alone from day 6 post treatment to day 12 post treatment. The combination therapy also normalized the hematological parameters of infected mice. Conclusions: An oral dose of pyronaridine tetraphosphate combined with a subcutaneous dose of diminazene aceturate inhibits Babesia in vitro and in mice, suggesting it might be a new paradigm for the treatment of babesiosis.

Diminazene aceturate or losartan ameliorates the functional, radiological and histopathological alterations in knee osteoarthritis rodent model: repurposing of the ACE2/Ang1-7/MasR cascade.

PURPOSE: Current therapies for osteoarthritis (OA) are limited to analgesics and anti-inflammatory drugs. Considering the importance of oxidative stress and inflammatory mediators in OA etiology, we tested the hypothesis that targeting the renin-angiotensin-aldosterone system (RAAS) can improve OA anomalies. Diminazene (DIZE), an activator of angiotensin-converting enzyme 2 and the angiotensin 2 type-1 receptor blocker losartan (LOS) were used for this purpose. METHODS: OA was induced by a single intra-articular injection of monosodium iodoacetate. The effects of exposure to DIZE or LOS for 21 days on OA anomalies in rats' knees were investigated. Evaluation of motor function, nociception, and inflammatory response was done using rotarod, knee bend and knee swelling tests. Markers of knee joint inflammation, and cellular oxidation in addition to the RAAS biomarkers, were assessed in knee tissues, along with radiological and histopathological investigations. RESULTS: Elevations in inflammatory and oxidative markers in knee tissues of OA rats were mostly improved by the two therapeutic drugs. Such effect was also reflected in the rotarod, knee bend and knee swelling tests. Treatment with DIZE has shown a more prominent effect than LOS in controlling OA-associated inflammation and cellular oxidation. Markers of RAAS have also shown better responsiveness to DIZE over LOS. CONCLUSIONS: DIZE has shown a prominent increase in the angiotensin 1-7 amount, highlighting the involvement of the signaling pathway in the immunomodulatory effect. The radiological and histopathology examination came to confirm the outcome of biochemical markers, nominating diminazene aceturate as a possible therapeutic option for OA.

Hyaluronic acid/diminazene aceturate combination ameliorates osteoarthritic anomalies in a rodent model: a role of the ACE2/Ang1-7/MasR axis.

The implication of the tissue-localized renin-angiotensin system (RAS) in the pathogenesis of osteoarthritis (OA) has been documented in the last decades. A combination of intraarticular (IA) corticosteroid and hyaluronic acid (HYAL) is approved for pain relief in patients with mild to moderate OA. Combining HYAL with an activator of angiotensin-converting enzyme 2, diminazen aceturate (DIZE), was evaluated in this study for its therapeutic potential. Monosodium iodoacetate was used to induce OA. The effects of daily administration of DIZE versus once-per-week IA injection of HYAL and a combination of both drugs for 21 days on OA deformities in rats' knees were observed. Evaluation of motor activities, pain, and inflammatory response was done using rotarod, knee bend, and knee swelling tests. RAS components, inflammatory biomarkers, and oxidative stress mediators were measured in the knee joint. X-ray radiological examination and histopathological investigations were used to assess joint degeneration and regeneration. Levels of both inflammatory and oxidative markers in knee joint homogenate of OA rats rose, and these increments were mostly improved by the three therapies with a more prominent effect of the drug combination, an effect that was also reflected in the behavioral tests. RAS markers have shown better responsiveness to the combination therapy over both drugs individually, showing a pronounced increase in the angiotensin 1-7 amount. Both radiological and histopathology investigations came to confirm the biochemical results, nominating a combination of HYAL and DIZE as a possible therapeutic option for OA.

Evaluating the inhibitory effect of resveratrol on the multiplication of several Babesia species and Theileria equi on in vitro cultures, and Babesia microti in mice.

Introduction: Histone post-translational modification is one of the most studied factors influencing epigenetic regulation of protozoan parasite gene expression, which is mediated by histone deacetylases (KDACs) and acetyltransferases (KATs). Objective and methods: The present study investigated the role of resveratrol (RVT) as an activator of histone deacetylases in the control of various pathogenic Babesia sp. and Theileria equi in vitro, as well as B. microti infected mice in vivo using fluorescence assay. Its role in mitigating the side effects associated with the widely used antibabesial drugs diminazene aceturate (DA) and azithromycin (AZM) has also been investigated. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi and Theileria equi (T. equi) was significantly inhibited (P < 0.05) by RVT treatments. The estimated IC50 values revealed that RVT has the greatest inhibitory effects on B. bovis growth in vitro, with an IC50 value of 29.51 ± 2.46 µM. Reverse transcription PCR assay showed that such inhibitory activity might be attributed to resveratrol's stimulatory effect on B. bovis KDAC3 (BbKADC3) as well as its inhibitory effect on BbKATS. RVT causes a significant decrease (P < 0.05) in cardiac troponin T (cTnT) levels in heart tissue of B. microti- infected mice, thereby indicating that RVT may play a part in reducing the cardiotoxic effects of AZM. Resveratrol showed an additive effect with imidocarb dipropionate in vivo. Treatment of B. microti-infected mice with a combined 5 mg/kg RVT and 8.5 mg/kg ID resulted in an 81.55% inhibition at day 10 postinoculation (peak of parasitemia). Conclusion: Our data show that RVT is a promising antibabesial pharmacological candidate with therapeutic activities that could overcome the side effects of the currently used anti-Babesia medications.

The ACE2 activator diminazene aceturate ameliorates colitis by repairing the gut-vascular barrier in mice.

Alleviating vascular barrier injury improves colitis. Angiotensin converting enzyme 2/angiotensin 1-7/Mas receptor (ACE2/Ang1-7/MasR) axis-related drugs have various biological properties, such as inhibition of inflammation and fibrosis, but their role in improving the gut-vascular barrier (GVB) has rarely been reported. This study aims to investigate the effects of diminazene aceturate (DIZE), an ACE2 activator, on vascular barrier damage in colitis. Mice were randomly divided into three groups: control, dextran sulfate sodium salt (DSS), and DIZE+DSS. Mice in the DSS group drank DSS for 8 days starting on day 4. Mice in the DIZE+DSS group were pregavaged with DIZE for 3 days and then drank DSS for 8 days while continuing to be gavaged with DIZE for 4 days. Mice were euthanized and samples were collected on the last day. Injury to colonic structure and colonic microvasculature was assessed by visual observation and appropriate staining. DSS-induced colonic and microvascular pathological damage in mice was substantially reversed by DIZE treatment. Molecular pathways were investigated by Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme linked immunosorbent assay (ELISA). DSS treatment upregulated angiotensin converting enzyme (ACE), angiotensin type 1 receptor (AT1R) protein, pro-inflammatory cytokines and inhibited tight junction-related protein expression. DIZE treatment activated ACE2/MasR protein expression and reversed epithelial barrier damage and inflammatory infiltration during DSS injury. In addition, DIZE treatment inhibited vascular endothelial growth factor A/vascular endothelial growth factor receptor 2/proto-oncogene tyrosine-protein kinase Src (VEGFA/VEGFR2/Src) pathway activation and restored vascular adhesion-linker protein vascular endothelial cadherin (VE-cadherin) expression during DSS injury. In conclusion, DIZE treatment ameliorated colitis, which was associated with balancing the two axes of the renin-angiotensin system (RAS) and repairing the GVB injury.

Diminazene aceturate exacerbates renal fibrosis after unilateral ureteral obstruction in female mice.

BACKGROUND: Diminazene aceturate (DIZE), an angiotensin-converting enzyme 2 (ACE2) activator, exerts anti-inflammatory and antifibrotic effects in a variety of human chronic diseases. However, the role of DIZE in kidney fibrosis and the underlying mechanism remain unclear. Therefore, we investigated the effects of DIZE on the progression of renal fibrosis after unilateral ureteral obstruction (UUO), a well-established model of chronic kidney disease. METHODS: C57BL/6 female or male mice were subjected to right UUO. Mice received 15 mg/kg DIZE or vehicle (saline) daily. On the 7th day after UUO, kidneys were collected for analysis of renal fibrosis (α-smooth muscle actin, phosphorylated SMAD3, transforming growth factor (TGF)-ß, Masson's trichrome, and Sirius red staining), inflammation (macrophage infiltration, proinflammatory cytokines/ chemokines), apoptosis/necrotic cell death (TUNEL and periodic acid-Schiff staining), and ACE2 activity and messenger RNA (mRNA) expression. RESULTS: Treatment with DIZE exacerbated renal fibrosis by upregulating the profibrotic TGF-ß/SMAD3 pathway, proinflammatory cytokine/chemokines (interleukin [IL]-1ß, monocyte chemoattractant protein-1, IL-6, and macrophage inflammatory protein-2) levels, M2 macrophage accumulation (CD206, IL-4, IL-10, and CX3CL1), and apoptotic/necrotic cell death in the obstructed kidneys of female mice but not male mice. However, DIZE treatment had no effect on ACE2 activity or mRNA expression. CONCLUSION: DIZE exacerbates UUO-induced renal fibrosis by aggravating tubular damage, apoptosis, and inflammation through independent of angiotensin (1-7), angiotensin II levels, and ACE2 expression/activity, rather than protecting against renal fibrosis after UUO. DIZE also has powerful effects on recruiting macrophages, including the M2-polarized subtype, in female UUO mice.

Pharmacologic Activation of Angiotensin-Converting Enzyme II Alleviates Diabetic Cardiomyopathy in db/db Mice by Reducing Reactive Oxidative Stress.

BACKGRUOUND: Diabetes mellitus is one of the most common chronic diseases worldwide, and cardiovascular disease is the leading cause of morbidity and mortality in diabetic patients. Diabetic cardiomyopathy (DCM) is a phenomenon characterized by a deterioration in cardiac function and structure, independent of vascular complications. Among many possible causes, the renin-angiotensin-aldosterone system and angiotensin II have been proposed as major drivers of DCM development. In the current study, we aimed to investigate the effects of pharmacological activation of angiotensin-converting enzyme 2 (ACE2) on DCM. METHODS: The ACE2 activator diminazene aceturate (DIZE) was administered intraperitoneally to male db/db mice (8 weeks old) for 8 weeks. Transthoracic echocardiography was used to assess cardiac mass and function in mice. Cardiac structure and fibrotic changes were examined using histology and immunohistochemistry. Gene and protein expression levels were examined using quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Additionally, RNA sequencing was performed to investigate the underlying mechanisms of the effects of DIZE and identify novel potential therapeutic targets for DCM. RESULTS: Echocardiography revealed that in DCM, the administration of DIZE significantly improved cardiac function as well as reduced cardiac hypertrophy and fibrosis. Transcriptome analysis revealed that DIZE treatment suppresses oxidative stress and several pathways related to cardiac hypertrophy. CONCLUSION: DIZE prevented the diabetes mellitus-mediated structural and functional deterioration of mouse hearts. Our findings suggest that the pharmacological activation of ACE2 could be a novel treatment strategy for DCM.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - A new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 hours in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5 %, 32 %, and 4.5 % more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - a new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 h in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5, 32, and 4.5% more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

Role of hormones in bone remodeling in the craniofacial complex: A review.

Background: Diseases such as periodontitis and osteoporosis are expected to rise tremendously by 2050. Bone formation and remodeling are complex processes that are disturbed in a variety of diseases influenced by various hormones. Objective: This study aimed to review and present the roles of various hormones that regulate bone remodeling of the craniofacial complex. Methods: A literature search was conducted on PubMed and Google Scholar for studies related to hormones and jawbone. Search strategies included the combinations ("name of hormone" + "dental term") of the following terms: "hormones", "oxytocin", "estrogen", "adiponectin", "parathyroid hormone", "testosterone", "insulin", "angiotensin", "cortisol", and "erythropoietin", combined with a dental term "jaw bone", "alveolar bone", "dental implant", "jaw + bone regeneration, healing or repair", "dentistry", "periodontitis", "dry socket", "osteoporosis" or "alveolitis". The papers were screened according to the inclusion criteria from January 1, 2000 to March 31, 2021 in English. Publications included reviews, book chapters, and original research papers; in vitro studies, in vivo animal, or human studies, including clinical studies, and meta-analyses. Results: Bone formation and remodeling is a complex continuous process involving many hormones. Bone volume reduction following tooth extractions and bone diseases, such as periodontitis and osteoporosis, cause serious problems and require a great understanding of the process. Conclusion: Hormones are with us all the time, shape our development and regulate homeostasis. Newly discovered effects of hormones influencing bone healing open the possibilities of using hormones as therapeutics to combat bone-related diseases.

Repurposing streptomycin and chloramphenicol against bacterial pathogens by combination with diminazene aceturate.

Bacterial resistance is a threat to health worldwide, mainly due to reduced effective treatment. In this context, the search for strategies to control such infections and suppress antimicrobial resistance is necessary. One of the strategies that has been used is combination therapy. In the present work, we investigated the in vitro efficacy of the antimicrobials diminazene aceturate (DA), chloramphenicol (CHL), and streptomycin (STP) alone and in combination against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus clinical isolates. DA was capable of inhibiting all strains with MIC of 25-400 µg mL-1, while STP and CHL showed antibacterial activity with minimum inhibitory concentration (MICs) of ≤3.12-400 µg mL-1. The combination of aceturate with STP showed synergism toward almost all Gram-negative bacteria, with fractional inhibitory concentration index (FICIs) of 0.09-0.37. In addition, for CHL and aceturate, synergisms for Gram-negative and -positive strains were observed. A time-kill assay against E. coli revealed that the aceturate and STP combination can inhibit bacterial growth in a shorter time when compared with single antibiotics. In addition, antimicrobials did not show hemolytic activity even at the highest concentrations used. Therefore, the antimicrobial combinations presented in this work showed important results, demonstrating that combined therapy can be used as an alternative strategy for pathogen control.

African trypanosome infection patterns in cattle in a farm setting in Southern Ghana.

African Trypanosomiasis is a debilitating disease in both humans and animals that occurs in sub-Saharan Africa and has a severe negative impact on the livelihood of people in the affected areas. The disease is caused by protozoan parasites of the genus Trypanosoma, which is often described simply as blood-borne; however, a number of studies have shown the parasite inhabits many different environments within the host. Control of the disease involves measures that include the use of trypanocidal drugs to which there are growing number of reported cases of resistance. Here, the patterns of trypanosome DNA presence during a diminazene aceturate treatment round on a cohort of cattle in Adidome, Ghana were assessed. A group of 24 cows were selected irrespective of age and sex and the infecting trypanosome species followed for 18 days before and after treatment with diminazene aceturate in the blood and skin of the animals using a diagnostic nested PCR that targeted the alpha-beta tubulin gene array. Persistence of trypanosome DNA was observed over the period and parasite DNA was readily detected in both the skin and blood, with parasite DNA disappearing and reappearing in both across the study. Moreover, there was limited correlation between the parasite DNA detected in the skin and blood. Overall, the data show the patterns of a natural trypanosome infection during drug treatment. In addition, the diagnostic potential of sampling the skin for African trypanosomiasis is highlighted.

Diminazene aceturate uses different pathways to induce relaxation in healthy and atherogenic blood vessels.

Diminazene aceturate (DIZE), a putative angiotensin-converting enzyme 2 (ACE2) activator, elicits relaxation in various animal models. This study aimed to determine the relaxing mechanisms in internal iliac arteries utilised by DIZE in healthy and atherogenic rabbit models. Studies were conducted on internal iliac artery rings retrieved from male New Zealand White rabbits fed a 4-week healthy control (n = 24) or atherogenic diet (n = 20). To investigate pathways utilised by DIZE to promote arterial relaxation, a DIZE dose response [10-9.0 M - 10-5.0 M] was performed on pre-contracted rings incubated with pharmaceuticals that target: components of the renin-angiotensin system; endothelial- and vascular smooth muscle-dependent mechanisms; protein kinases; and potassium channels. ACE2 expression was quantified by immunohistochemistry analysis following a 2 hr or 4 hr DIZE incubation. DIZE significantly enhanced vessel relaxation in atherogenic rings at doses [10-5.5 M] (p < 0.01) and [10-5.0 M] (p < 0.0001), when compared to healthy controls. Comprehensive results from functional isometric studies determined that DIZE causes relaxation via different mechanisms depending on pathology. For the first time, we report that in healthy blood vessels DIZE exerts its direct relaxing effect through ACE2/AT2R and NO/sGC pathways; however, in atherogenesis this switches to MasR, arachidonic acid pathway (i.e., COX1/2, EET and DHET), MCLP, Ca2+ activated voltage channels, AMPK and ERK1/2. Moreover, quantitative immunohistochemical analysis demonstrated that DIZE increases artery ACE2 expression in a time dependent manner. We provide a detailed investigation of DIZE's mechanisms and demonstrate for the first time that in healthy and atherogenic arteries DIZE provides beneficial effects through directly inducing relaxation, albeit via different pathways.

DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells.

The protective arm of the renin-angiotensin system (RAS), the ACE 2/Ang-(1-7)/MasR axis, has become a new anti-inflammatory target. As a specific activator of ACE2, diminazene aceturate (DA) can promote anti-inflammatory effects by regulating the ACE2/Ang-(1-7)/MasR axis. However, due to the reported toxicity of DA, its application has been limited. In the current study, we synthesized a low toxicity DA derivative 3 (DAD3) and sought to determine whether DAD3 can also activate ACE2 in bovine mammary epithelial cells (BMEC) and regulate the RAS system to inhibit inflammation. We found that both DA and DAD3 can activate and promote ACE2 expression in BMEC. iRNA-mediated knockdown of ACE2 demonstrated that DAD3 activates the ACE2/Ang-(1-7)/MasR axis and plays an anti-inflammatory role in BMEC. Furthermore, the inhibitory effects of DA and DAD3 on the protein phosphorylation of MAPK and NF-κB pathways were reduced in ACE2-silenced BMEC. Our findings show that ACE2 is a target of DAD3, which leads to inhibition of the MAPK and NF-κB signalling pathways and protects against LPS-induced inflammation in BMEC. Thus, DAD3 may provide a new strategy to treat dairy cow mastitis.

Antiviral potential of diminazene aceturate against SARS-CoV-2 proteases using computational and in vitro approaches.

Diminazene aceturate (DIZE), an antiparasitic, is an ACE2 activator, and studies show that activators of this enzyme may be beneficial for COVID-19, disease caused by SARS-CoV-2. Thus, the objective was to evaluate the in silico and in vitro affinity of diminazene aceturate against molecular targets of SARS-CoV-2. 3D structures from DIZE and the proteases from SARS-CoV-2, obtained through the Protein Data Bank and Drug Database (Drubank), and processed in computer programs like AutodockTools, LigPlot, Pymol for molecular docking and visualization and GROMACS was used to perform molecular dynamics. The results demonstrate that DIZE could interact with all tested targets, and the best binding energies were obtained from the interaction of Protein S (closed conformation -7.87 kcal/mol) and Mpro (-6.23 kcal/mol), indicating that it can act both by preventing entry and viral replication. The results of molecular dynamics demonstrate that DIZE was able to promote a change in stability at the cleavage sites between S1 and S2, which could prevent binding to ACE2 and fusion with the membrane. In addition, in vitro tests confirm the in silico results showing that DIZE could inhibit the binding between the spike receptor-binding domain protein and ACE2, which could promote a reduction in the virus infection. However, tests in other experimental models with in vivo approaches are needed.

Klotho restoration via ACE2 activation: A potential therapeutic strategy against acute kidney injury-diabetes comorbidity.

Acute kidney injury (AKI) is a collection of clinical syndromes with persistent increases in morbidity and mortality rates. Hyperglycemia is a risk factor for AKI development. Renin-angiotensin-aldosterone system (RAS) disequilibrium and Klotho downregulation also play a pivotal role in the pathogenesis of AKI. Moreover, the relationship between Klotho and ACE2 (a component of non-conventional RAS) regulation in AKI remains an unexplored area of research. Hence, in this study, we investigated ACE2 and Klotho regulation in AKI using ischemic Wistar rats and NRK52E cells under normal and hyperglycemic conditions. Our findings suggested that hyperglycemia exacerbates renal ischemia-reperfusion injury (IRI)/hypoxia-reperfusion injury (HRI) induced AKI. Systemic and renal Klotho deficiency is a novel hallmark of AKI. Additionally, ACE2 is a protective component of the RAS, and its inhibition/deficiency leads to inflammation, apoptosis, Klotho downregulation, and thus AKI development. However, ACE2 activation resulted in the amelioration of AKI. Importantly, ACE2 plays an important role in Klotho upregulation, which might act as an intermediate for ACE2-mediated reno-protection. In conclusion, ACE2 activator i.e. DIZE restored endogenous ACE2-Ang-(1-7)-Klotho level, inhibited apoptosis and inflammation, and ameliorates IRI/HRI induced AKI under diabetic and non-diabetic conditions. Hence, in future, targeting ACE2-Ang-(1-7)-Klotho axis may prove a novel therapeutic strategy against AKI, where further preclinical and clinical investigations are required to verify the clinical potential of this finding.

Diminazene Aceturate Reduces Angiotensin II Constriction and Interacts with the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

Diminazene aceturate (DIZE) is a putative angiotensin-converting enzyme 2 (ACE2) activator and angiotensin type 1 receptor antagonist (AT1R). Its simple chemical structure possesses a negatively charged triazene segment that is homologous to the tetrazole of angiotensin receptor blockers (ARB), which explains its AT1R antagonistic activity. Additionally, the activation of ACE2 by DIZE converts the toxic octapeptide angiotensin II (AngII) to the heptapeptides angiotensin 1-7 and alamandine, which promote vasodilation and maintains homeostatic balance. Due to DIZE's protective cardiovascular and pulmonary effects and its ability to target ACE2 (the predominant receptor utilized by severe acute respiratory syndrome coronavirus 2 to enter host cells), it is a promising treatment for coronavirus 2019 (COVID-19). To determine DIZE's ability to inhibit AngII constriction, in vitro isometric tension analysis was conducted on rabbit iliac arteries incubated with DIZE or candesartan and constricted with cumulative doses of AngII. In silico docking and ligand interaction studies were performed to investigate potential interactions between DIZE and other ARBs with AT1R and the spike protein/ACE2 complex. DIZE, similar to the other ARBs investigated, was able to abolish vasoconstriction in response to AngII and exhibited a binding affinity for the spike protein/ACE2 complex (PDB 6LZ6). These results support the potential of DIZE as a treatment for COVID-19.