Mitochondrial fatty acid beta-oxidation: a possible therapeutic target for skeletal muscle lipotoxicity in peripheral artery disease myopathy.

Peripheral artery disease (PAD) is an atherosclerotic disease impacting over 200 million individuals and the prevalence increases with age. PAD occurs when plaque builds up within the peripheral arteries, leading to reduced blood flow and oxygen supply to the outer extremities. Individuals who experience PAD suffer from ischemia, which is typically accompanied by significant damage to skeletal muscles. Additionally, this tissue damage affects mitochondria, causing them to become dysregulated and dysfunctional, resulting in decreased metabolic rates. As there is no known cure for PAD, researchers are exploring potential therapeutic targets by examining coexisting cardiovascular conditions and metabolic risk factors, such as the aging process. Among these comorbidities, type-two diabetes mellitus and obesity are particularly common in PAD cases. These conditions, along with aging itself, are associated with an elevated accumulation of ectopic lipids within skeletal muscles, similar to what is observed in PAD. Researchers have attempted to reduce excess lipid accumulation by increasing the rate of fatty acid beta oxidation. Manipulating acetyl coenzyme A carboxylase 2, a key regulatory protein of fatty acid beta oxidation, has been the primary focus of such research. When acetyl coenzyme A carboxylase 2 is inhibited, it interrupts the conversion of acetyl-CoA into malonyl-CoA, resulting in an increase in the rate of fatty acid beta oxidation. By utilizing samples from PAD patients and applying the pharmacological strategies developed for acetyl coenzyme A carboxylase 2 in diabetes and obesity to PAD, a potential new therapeutic avenue may emerge, offering hope for improved quality of life for individuals suffering from PAD.

PCSK9 inhibition attenuates alcohol-associated neuronal oxidative stress and cellular injury.

Alcohol Use Disorder (AUD) is a persistent condition linked to neuroinflammation, neuronal oxidative stress, and neurodegenerative processes. While the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has demonstrated effectiveness in reducing liver inflammation associated with alcohol, its impact on the brain remains largely unexplored. This study aimed to assess the effects of alirocumab, a monoclonal antibody targeting PCSK9 to lower systemic low-density lipoprotein cholesterol (LDL-C), on central nervous system (CNS) pathology in a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for six weeks in 32 male rats subjected to a 35 % ethanol liquid diet or a control liquid diet (n = 8 per group). The study evaluated PCSK9 expression, LDL receptor (LDLR) expression, oxidative stress, and neuroinflammatory markers in brain tissues. Chronic ethanol exposure increased PCSK9 expression in the brain, while alirocumab treatment significantly upregulated neuronal LDLR and reduced oxidative stress in neurons and brain vasculature (3-NT, p22phox). Alirocumab also mitigated ethanol-induced microglia recruitment in the cortex and hippocampus (Iba1). Additionally, alirocumab decreased the expression of pro-inflammatory cytokines and chemokines (TNF, CCL2, CXCL3) in whole brain tissue and attenuated the upregulation of adhesion molecules in brain vasculature (ICAM1, VCAM1, eSelectin). This study presents novel evidence that alirocumab diminishes oxidative stress and modifies neuroimmune interactions in the brain elicited by chronic ethanol exposure. Further investigation is needed to elucidate the mechanisms by which PCSK9 signaling influences the brain in the context of chronic ethanol exposure.

NASH triggers cardiometabolic HFpEF in aging mice.

Both heart failure with preserved ejection fraction (HFpEF) and non-alcoholic fatty liver disease (NAFLD) develop due to metabolic dysregulation, has similar risk factors (e.g., insulin resistance, systemic inflammation) and are unresolved clinical challenges. Therefore, the potential link between the two disease is important to study. We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age dependent effects of NASH on cardiac function. C57Bl/6 J middle aged (10 months old) and aged mice (24 months old) were fed either control or choline deficient (CDAA) diet for 8 weeks. Before termination, echocardiography was performed. Upon termination, organ samples were isolated for histological and molecular analysis. CDAA diet led to the development of NASH in both age groups, without inducing weight gain, allowing to study the direct effect of NASH on cardiac function. Mice with NASH developed hepatomegaly, fibrosis, and inflammation. Aged animals had increased heart weight. Conventional echocardiography revealed normal systolic function in all cohorts, while increased left ventricular volumes in aged mice. Two-dimensional speckle tracking echocardiography showed subtle systolic and diastolic deterioration in aged mice with NASH. Histologic analyses of cardiac samples showed increased cross-sectional area, pronounced fibrosis and Col1a1 gene expression, and elevated intracardiac CD68+ macrophage count with increased Il1b expression. Conventional echocardiography failed to reveal subtle change in myocardial function; however, 2D speckle tracking echocardiography was able to identify diastolic deterioration. NASH had greater impact on aged animals resulting in cardiac hypertrophy, fibrosis, and inflammation.

EVALUATION OF COMPONENTS OF THE EXTRACELLULAR PURINERGIC SIGNALING SYSTEM IN HUMAN SEPSIS.

ABSTRACT: Objective: Extracellular purines such as adenosine triphosphate (ATP), uridine triphosphate (UTP), and uridine diphosphate (UDP) and the ATP degradation product adenosine are biologically active signaling molecules, which accumulate at sites of metabolic stress in sepsis. They have potent immunomodulatory effects by binding to and activating P1 or adenosine and P2 receptors on the surface of leukocytes. Here we assessed the levels of extracellular purines, their receptors, metabolic enzymes, and cellular transporters in leukocytes of septic patients. Methods: Peripheral blood mononuclear cells (PBMCs), neutrophils, and plasma were isolated from blood obtained from septic patients and healthy control subjects. Ribonucleic acid was isolated from cells, and mRNA levels for purinergic receptors, enzymes, and transporters were measured. Adenosine triphosphate, UTP, UDP, and adenosine levels were evaluated in plasma. Results: Adenosine triphosphate levels were lower in septic patients than in healthy individuals, and levels of the other purines were comparable between the two groups. Levels of P1 and P2 receptors did not differ between the two patient groups. mRNA levels of ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1 or CD39 increased, whereas those of NTPDase2, 3, and 8 decreased in PBMCs of septic patients when compared with healthy controls. CD73 mRNA was lower in PBMCs of septic than in healthy individuals. Equilibrative nucleoside transporter (ENT) 1 mRNA concentrations were higher and ENT2, 3, and 4 mRNA concentrations were lower in PBMCs of septic subjects when compared with healthy subjects. Concentrative nucleoside transporter (CNT) 1 mRNA levels were higher in PBMCs of septic versus healthy subjects, whereas the mRNA levels of CNT2, 3, and 4 did not differ. We failed to detect differences in mRNA levels of purinergic receptors, enzymes, and transporters in neutrophils of septic versus healthy subjects. Conclusion: Because CD39 degrades ATP to adenosine monophosphate (AMP), the lower ATP levels in septic individuals may be the result of increased CD39 expression. This increased degradation of ATP did not lead to increased adenosine levels, which may be explained by the decreased expression of CD73, which converts AMP to adenosine. Altogether, our results demonstrate differential regulation of components of the purinergic system in PBMCs during human sepsis.

PCSK9, A Promising Novel Target for Age-Related Cardiovascular Dysfunction.

Cardiovascular diseases (CVDs) are the leading cause of death among elderly people. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important regulator of cholesterol metabolism. Herein, we investigated the role of PCSK9 in age-related CVD. Both in humans and rats, blood PCSK9 level correlated positively with increasing age and the development of cardiovascular dysfunction. Age-related fatty degeneration of liver tissue positively correlated with serum PCSK9 levels in the rat model, while development of age-related nonalcoholic fatty liver disease correlated with cardiovascular functional impairment. Network analysis identified PCSK9 as an important factor in age-associated lipid alterations and it correlated positively with intima-media thickness, a clinical parameter of CVD risk. PCSK9 inhibition with alirocumab effectively reduced the CVD progression in aging rats, suggesting that PCSK9 plays an important role in cardiovascular aging.

A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence.

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

Data-driven transcriptomics analysis identifies PCSK9 as a novel key regulator in liver aging.

The liver, as a crucial metabolic organ, undergoes significant pathological changes during the aging process, which can have a profound impact on overall health. To gain a comprehensive understanding of these alterations, we employed data-driven approaches, along with biochemical methods, histology, and immunohistochemistry techniques, to systematically investigate the effects of aging on the liver. Our study utilized a well-established rat aging model provided by the National Institute of Aging. Systems biology approaches were used to analyze genome-wide transcriptomics data from liver samples obtained from young (4-5 months old) and aging (20-21 months old) Fischer 344 rats. Our findings revealed pathological changes occurring in various essential biological processes in aging livers. These included mitochondrial dysfunction, increased oxidative/nitrative stress, decreased NAD + content, impaired amino acid and protein synthesis, heightened inflammation, disrupted lipid metabolism, enhanced apoptosis, senescence, and fibrosis. These results were validated using independent datasets from both human and rat aging studies. Furthermore, by employing co-expression network analysis, we identified novel driver genes responsible for liver aging, confirmed our findings in human aging subjects, and pointed out the cellular localization of the driver genes using single-cell RNA-sequencing human data. Our study led to the discovery and validation of a liver-specific gene, proprotein convertase subtilisin/kexin type 9 (PCSK9), as a potential therapeutic target for mitigating the pathological processes associated with aging in the liver. This finding envisions new possibilities for developing interventions aimed to improve liver health during the aging process.

Elimination of senescent cells by treatment with Navitoclax/ABT263 reverses whole brain irradiation-induced blood-brain barrier disruption in the mouse brain.

Whole brain irradiation (WBI), a commonly employed therapy for multiple brain metastases and as a prophylactic measure after cerebral metastasis resection, is associated with a progressive decline in neurocognitive function, significantly impacting the quality of life for approximately half of the surviving patients. Recent preclinical investigations have shed light on the multifaceted cerebrovascular injury mechanisms underlying this side effect of WBI. In this study, we aimed to test the hypothesis that WBI induces endothelial senescence, contributing to chronic disruption of the blood-brain barrier (BBB) and microvascular rarefaction. To accomplish this, we utilized transgenic p16-3MR mice, which enable the identification and selective elimination of senescent cells. These mice were subjected to a clinically relevant fractionated WBI protocol (5 Gy twice weekly for 4 weeks), and cranial windows were applied to both WBI-treated and control mice. Quantitative assessment of BBB permeability and capillary density was performed using two-photon microscopy at the 6-month post-irradiation time point. The presence of senescent microvascular endothelial cells was assessed by imaging flow cytometry, immunolabeling, and single-cell RNA-sequencing (scRNA-seq). WBI induced endothelial senescence, which associated with chronic BBB disruption and a trend for decreased microvascular density in the mouse cortex. In order to investigate the cause-and-effect relationship between WBI-induced senescence and microvascular injury, senescent cells were selectively removed from animals subjected to WBI treatment using Navitoclax/ABT263, a well-known senolytic drug. This intervention was carried out at the 3-month post-WBI time point. In WBI-treated mice, Navitoclax/ABT263 effectively eliminated senescent endothelial cells, which was associated with decreased BBB permeability and a trend for increased cortical capillarization. Our findings provide additional preclinical evidence that senolytic treatment approaches may be developed for prevention of the side effects of WBI.

Adenosine metabolized from extracellular ATP ameliorates organ injury by triggering A2BR signaling.

BACKGROUND: Trauma and a subsequent hemorrhagic shock (T/HS) result in insufficient oxygen delivery to tissues and multiple organ failure. Extracellular adenosine, which is a product of the extracellular degradation of adenosine 5' triphosphate (ATP) by the membrane-embedded enzymes CD39 and CD73, is organ protective, as it participates in signaling pathways, which promote cell survival and suppress inflammation through adenosine receptors including the A2BR. The aim of this study was to evaluate the role of CD39 and CD73 delivering adenosine to A2BRs in regulating the host's response to T/HS. METHODS: T/HS shock was induced by blood withdrawal from the femoral artery in wild-type, global knockout (CD39, CD73, A2BR) and conditional knockout (intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl) mice. At 3 three hours after resuscitation, blood and tissue samples were collected to analyze organ injury. RESULTS: T/HS upregulated the expression of CD39, CD73, and the A2BR in organs. ATP and adenosine levels increased after T/HS in bronchoalveolar lavage fluid. CD39, CD73, and A2BR mimics/agonists alleviated lung and liver injury. Antagonists or the CD39, CD73, and A2BR knockout (KO) exacerbated lung injury, inflammatory cytokines, and chemokines as well as macrophage and neutrophil infiltration and accumulation in the lung. Agonists reduced the levels of the liver enzymes aspartate transferase and alanine transaminase in the blood, whereas antagonist administration or CD39, CD73, and A2BR KO enhanced enzyme levels. In addition, intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl mice showed increased intestinal injury compared to their wild-type VillinCre controls. CONCLUSION: In conclusion, the CD39-CD73-A2BR axis protects against T/HS-induced multiple organ failure.

Adenosine signaling as target in cardiovascular pharmacology.

Increasing evidence demonstrated the relevance of adenosine system in the onset and development of cardiovascular diseases, such as hypertension, myocardial infarct, ischemia, hypertension, heart failure, and atherosclerosis. In this regard, intense research efforts are being focused on the characterization of the pathophysiological significance of adenosine, acting at its membrane receptors named A1, A2A, A2B, and A3 receptors, in cardiovascular diseases. The present review article provides an integrated and comprehensive overview about current clinical and pre-clinical evidence about the role of adenosine in the pathophysiology of cardiovascular diseases. Particular attention has been focused on current scientific evidence about the pharmacological ligands acting on adenosine pathway as useful tools to manage cardiovascular diseases.

Structural basis of selective cannabinoid CB2 receptor activation.

Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.

Cardiac Plin5 interacts with SERCA2 and promotes calcium handling and cardiomyocyte contractility.

The adult heart develops hypertrophy to reduce ventricular wall stress and maintain cardiac function in response to an increased workload. Although pathological hypertrophy generally progresses to heart failure, physiological hypertrophy may be cardioprotective. Cardiac-specific overexpression of the lipid-droplet protein perilipin 5 (Plin5) promotes cardiac hypertrophy, but it is unclear whether this response is beneficial. We analyzed RNA-sequencing data from human left ventricle and showed that cardiac PLIN5 expression correlates with up-regulation of cardiac contraction-related processes. To investigate how elevated cardiac Plin5 levels affect cardiac contractility, we generated mice with cardiac-specific overexpression of Plin5 (MHC-Plin5 mice). These mice displayed increased left ventricular mass and cardiomyocyte size but preserved heart function. Quantitative proteomics identified sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) as a Plin5-interacting protein. In situ proximity ligation assay further confirmed the Plin5/SERCA2 interaction. Live imaging showed increases in intracellular Ca2+ release during contraction, Ca2+ removal during relaxation, and SERCA2 function in MHC-Plin5 versus WT cardiomyocytes. These results identify a role of Plin5 in improving cardiac contractility through enhanced Ca2+ signaling.

A 2A ADENOSINE RECEPTORS REGULATE MULTIPLE ORGAN FAILURE AFTER HEMORRHAGIC SHOCK IN MICE.

ABSTRACT: Trauma hemorrhagic shock (T/HS) is a clinical condition that causes multiple organ failure that needs rapid intervention. Restricted oxygen at the cellular level causes inflammation and subsequent cell death. Adenosine triphosphate is the universal intracellular energy currency and an important extracellular inflammatory signaling molecule. Adenosine, an endogenous nucleotide formed as a result of the breakdown of adenosine triphosphate, is also released during T/HS. Adenosine binds to four G protein-coupled receptors (A 1R , A 2a , A 2b , A 3R ) called adenosine receptors or P1 receptors. In the present study, we evaluated the effect of activation, inactivation, and genetic absence of A2aR (A2aR -/- mice) on T/HS-induced multiple organ failure. Wild-type mice were pretreated (30 min before shock induction) with an agonist or antagonist and then subjected to T/HS by withdrawing arterial blood and maintaining the blood pressure between 28 and 32 mm Hg. A2aR -/- mice were subjected to T/HS in the absence of pharmacologic treatment. Neutrophil sequestration was assessed by detecting myeloperoxidase, and Evans blue dye (EBD) method was used to analyze lung permeability. Blood and lung inflammatory cytokine levels were determined by sandwich enzyme-linked immunosorbent assay. The liver enzymes aspartate aminotransferase and alanine aminotransferase were determined spectrophotometrically from plasma. Activation of the apoptotic cascade was evaluated using a mouse apoptosis array. Our results demonstrate that the selective A2aR agonist CGS21680 decreases lung neutrophil sequestration, lung proinflammatory cytokines IL-6 and TNF-α, and bronchoalveolar lavage EBD. Pretreatment with the selective antagonist ZM241385 and genetic blockade in A2aR -/- mice increased neutrophil sequestration, proinflammatory cytokine levels, and bronchoalveolar lavage fluid EBD. The myeloperoxidase level in the lung was also increased in A2aR -/- mice. We observed that antiapoptotic markers decreased significantly with the absence of A2aR in the lung and spleen after T/HS. In conclusion, our data demonstrate that activation of A2aR regulates organ injury and apoptosis in the setting of T/HS.

A2A adenosine receptor activation prevents neutrophil aging and promotes polarization from N1 towards N2 phenotype.

Extracellular adenosine is a biologically active signaling molecule that accumulates at sites of metabolic stress in sepsis. Extracellular adenosine has potent immunosuppressive effects by binding to and activating G protein-coupled A2A adenosine receptors (A2AARs) on the surface of neutrophils. A2AAR signaling reproduces many of the phenotypic changes in neutrophils that are characteristic of sepsis, including decreased degranulation, impaired chemotaxis, and diminished ability to ingest and kill bacteria. We hypothesized that A2AARs also suppress neutrophil aging, which precedes cell death, and N1 to N2 polarization. Using human neutrophils isolated from healthy subjects, we demonstrate that A2AAR stimulation slows neutrophil aging, suppresses cell death, and promotes the polarization of neutrophils from an N1 to N2 phenotype. Using genetic knockout and pharmacological blockade, we confirmed that A2AARs decrease neutrophil aging in murine sepsis induced by cecal ligation and puncture. A2AARs expression is increased in neutrophils from septic patients compared to healthy subject but A2AAR expression fails to correlate with aging or N1/N2 polarization. Our data reveals that A2AARs regulate neutrophil aging in healthy but not septic neutrophils.

Maternal binge alcohol consumption leads to distinctive acute perturbations in embryonic cardiac gene expression profiles.

BACKGROUND: Excessive alcohol consumption during pregnancy is associated with high risk of congenital heart defects, but it is unclear how alcohol specifically affects heart development during the acute aftermath of a maternal binge drinking episode. We hypothesize that administration of a single maternal binge dose of alcohol to pregnant mice at embryonic day 9.5 (E9.5) causes perturbations in the expression patterns of specific genes in the developing heart in the acute period (1-3 days) following the binge episode. To test this hypothesis and identify strong candidate ethanol-sensitive target genes of interest, we adapted a mouse binge alcohol model that is associated with a high incidence of congenital heart defects as described below. METHODS/RESULTS: Pregnant mice were administered a single dose of alcohol (2.5 g/kg in saline) or control (saline alone) via oral gavage. To evaluate the impact of maternal binge alcohol on cardiac gene expression profiles, we isolated embryonic hearts from both groups (n = 5/group) at 24, 48, and 72 h post-gavage for transcriptomic analyses. RNA was extracted and evaluated using quantitative RNA-sequencing (RNA-Seq) methods. To identify a cohort of binge-altered cardiac genes, we set the threshold for change at >2.0-fold difference with adjusted p < 0.05 versus control.  RNA-Seq analysis of cardiac gene expression revealed that of the 17 genes that were altered within the first 48 h post-binge, with the largest category consisting of transcription factors (Alx1, Alx4, HoxB7, HoxD8, and Runx2), followed by signaling molecules (Adamts18, Dkk2, Rtl1, and Wnt7a). Furthermore, multiple comparative and pathway analyses suggested that several of the candidate genes identified through differential RNA-Seq analysis may interact through certain common pathways. To investigate this further, we performed gene-specific qPCR analyses for three representative candidate targets: Runx2, Wnt7a, and Mlxipl. Notably, only Wnt7a showed significantly (p < 0.05) decreased expression in response to maternal binge alcohol in the qPCR assays. CONCLUSIONS: These findings identify Wnt7a and a short list of potential other candidate genes and pathways for further study, which could provide mechanistic insights into how maternal binge alcohol consumption produces congenital cardiac malformations.

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.