bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of 4‐Methylumbelliferone (4‐MU) on Hyperglycemia in Type 2 Diabetic Mice
Authors: Edwards Jasmine Sheria , Sunkari Vivekananda , Bollyky Paul
Published: November 2023

Hyperglycemia is the underlying condition in diabetes that is associated with increase in morbidity and mortality. There is a need for other and more cost‐effective treatments directed at regulating blood‐glucose levels. Hyaluronan (HA) is an extracellular matrix component linked to diabetes. Higher concentrations of HA are found in the pancreatic islets and serum of individuals with Type 2 Diabetes (T2D). Based on these findings we hypothesized that inhibition of HA synthesis can lead to improved glycemic control in Type II Diabetics. To determine the effects of HA inhibition in T2D, 4‐Methylumbelliferone (4‐MU) was used to treat diabetic mice. Diabetic mice fed 4‐MU rapidly (<48 hours) became normoglycemic and maintained glycemic control through the duration of the experiment. These animals demonstrated a significant decrease in blood‐glucose and a partial decrease in weight. Quantification of insulin by enzyme‐linked immunosorbent assay (ELISA) showed that both serum and islets cultures from 4‐MU treated mice exhibited increased insulin production. Consistent with this data, mice lacking CD44 (HA receptor) show higher levels of insulin production while islets from CD44−/− mice produce more insulin in response to glucose challenge ex vivo. This data indicates that HA is increased in T2D and negatively regulates insulin production in a CD44‐dependent manner. We propose that 4‐MU, the active ingredient in a drug called “hymecromone” that is currently approved to treat biliary spasm, could be repurposed to treat hyperglycemia in T2D.

Support or Funding Information

This work was supported in part by National Institutes of Health grants and a BIRT supplement. This work was also supported by grants from the Juvenile Diabetes Research Foundation and Larry L Hillblom foundation fellowship.
Weight Management in Primary Care Validated by Evidence Based Algorithm
Authors: Jacks Denise G , Einstein George P , Tulp Orien L
Published: November 2023

To determine the efficacy of evidence based algorithms [EBA] applied to weight loss management in a primary setting, we examined four cross sectional correlational studies on weight loss and management in over 1,000 out‐patients. Patients encompassed a wide age spectrum [age 17 to 78 yrs], 70% females, Body Mass Index [BMI] 27–45 kg/m2. Patients with type II diabetes [NIDDM] were permitted if controlled with drugs or diet. Markers of metabolic syndrome [MS] including elevated blood pressure [BP], dyslipidemia [elevated triglycerides [TG], lowered HDL‐Cholesterol [HDL‐C], elevated fasting blood glucose [FBG], and central obesity [CO] were monitored for 52, 56, or 102 weeks using one of 4 separate drug or drug combinations [Lorcaserin, Phentermine/Topiramate, Naltrexone/Bupropion, Liraglutide]. Results indicated all groups lost weight [3.3 to 6.6 lbs] with corresponding decreases in waist circumference [−2.5 to 5.2 in], plasma TG [−4.8 to 13.3 mg/dl, Systolic [−0.7 to 2.8 mmHg.] and Diastolic BP [−0.8–7.9 mmHg] and FBG [−.0.8 to −7.9 mg/dl] with the greatest weight loss with the Phentermine/Topiramate combination after 52 to 102 weeks of observation. These results indicate that long term administration of weight loss medications can be a safe and effective regimen for weight control.

Support or Funding Information

Supported by Institutional Resources of USAT and the Einstein Institute
Mechanisms of Tolerance to delta‐9‐THC in Rodent Models of Pathological Pain
Authors: Morgan Daniel , Yuill Matthew , Zee Michael L , LaFleur Rebecca , Guindon Josee
Published: November 2023

The use of cannabinoids in pain management is of significant interest due to the antinociceptive efficacy of Δ9‐THC. However, tolerance to the antinociceptive effects of Δ9‐THC develops with repeated treatment. The objective of this study was to examine tolerance to daily administration of Δ9‐THC in clinically relevant rodent models of pathological pain. The effects of Δ9‐THC in pathological pain were assessed in male wild‐type and cannabinoid receptor 1 (CB1) desensitization‐resistant S426A/S430A mutant mice. First, we assessed tolerance to Δ9‐THC in mice subjected to the formalin test of inflammatory pain. Intraplantar formalin injection (10 μl at 2.5 %) produces a biphasic nociceptive response consisting of acute and inflammatory pain phases. Experimental groups were subjected to the formalin test after receiving daily intraperitoneal injections of Δ9‐THC (6 mg/kg) ranging from zero to twelve days. Wild‐type mice exhibited complete tolerance to the antinociceptive effects of Δ9‐THC in the formalin test after eight days of daily Δ9‐THC. Interestingly, we find that tolerance to the antinociceptive effects of Δ9‐THC is attenuated in S426A/S430A mutants for the inflammatory pain phase but not for the acute pain phase. The effect of the c‐jun N‐terminal kinase (JNK) inhibitor, SP600125, on Δ9‐THC tolerance was also examined. Pre‐treatment with SP600125 (3 mg/kg) attenuates tolerance for the antinociceptive effects of Δ9‐THC in both the acute and inflammatory pain phases. Tolerance to the anti‐allodynic effects of Δ9‐THC was examined in a mouse model of post‐surgical pain. Injection of Δ9‐THC completely alleviates thermal and mechanical allodynia following a 5 mm hindpaw surgical incision. Consistent with our previous work, tolerance to the anti‐allodynic effects of Δ9‐THC is attenuated in S426A/S430A mutant mice. These results demonstrate that Δ9‐THC exhibits robust antinociceptive effects in two separate rodent models of clinically relevant, pathological pain and are consistent with previous studies showing the attenuation of rapid tolerance to the antinociceptive effects of Δ9‐THC in S426A/S430A mutants using the tail‐flick and hotplate tests.

Support or Funding Information

Funded by NIH grants DA036385 (DJM), DA037355 (DJM), and Texas Tech University Health Sciences Center School of Medicine 121035 (JG).
Increases in Locomotor Activity during Spontaneous Cannabinoid Withdrawal in Mice
Authors: Bergman Jack A , Reingold Curtis G , Barkin Claire E , Bergman Nora R , Nikas Spyros P , Makriyannis Alex , Paronis Carol A.
Published: November 2023

Spontaneous cannabinoid (CB) withdrawal following exposure to Δ9‐tetrahydrocannabinol (THC) has been described man but has been inadequately characterized in mice. To address this issue, radiotelemetry was used to measure body temperature and locomotor activity in different groups of CD1 or C57 mice treated for 5–60 days with saline, 10–20 mg/kg/day of the CB partial agonist THC or 0.03–0.1 mg/kg/day of the CB high‐efficacy agonist AM2389. The effects of treatment were assessed throughout the regimen and for up to 10 days after the last injection to capture withdrawal‐related perturbations in thermoregulation or motor activity. Results show that acute exposure to AM2389 or THC decreased both temperature and activity. Rapid tolerance occurred to the hypothermic effects of the cannabinoids, whereas locomotor activity continued to be suppressed following each drug injection. In contrast, increases in locomotor activity were evident 12–72 hrs after discontinuing daily injections of AM2389, but not THC. Measurable effects following discontinuation of AM2389 injections were mild but reproducible across different groups of mice. These results indicate that the discontinuation of daily treatment with a CB high‐efficacy agonist will yield evidence of spontaneous withdrawal that may reflect prior dependence, but that the degree of cannabinoid dependence may vary in relation to the dose or efficacy of the agonist injected daily.

Support or Funding Information

Acknowledgements: Funded by NIH/NIDA: DA035411, DA031020
PPAR‐α Modulates The Anti‐Inflammatory Effect Of Melatonin In The Secondary Events Of Spinal Cord Injury
Authors: Paterniti Irene , Campolo Michela , Cordaro Marika , Impellizzeri Daniela , Siracusa Rosalba , Crupi Rosalia , Esposito Emanuela , Cuzzocrea Salvatore
Published: November 2023

Melatonin is the principal secretory product of the pineal gland and its role as an immumo‐modulator is well established. Recent evidence shows that melatonin is a scavenger of oxyradicals and peroxynitrite and reduces the development of inflammation and tissue injury events associated with spinal cord trauma. Previous results suggest that Peroxisome proliferator activated receptor alpha‐ α (PPAR‐α), an intracellular transcription factor activated by fatty acids, plays a role in control of secondary inflammatory process associated with spinal cord injury (SCI).

With the aim to characterize the role of PPAR‐α in melatonin mediated anti‐inflammatory activity, we tested the efficacy of melatonin (30 mg/kg) in an experimental model of spinal cord trauma induced in mice by the application of vascular clips (force of 24 g) to the dura via a four‐level T5‐T8 laminectomy, and comparing mice lacking PPAR‐α (PPAR‐αKO) with wild type (WT) mice.

The results obtained indicate that melatonin ‐mediated anti‐inflammatory activity is weakened in PPAR‐α KO mice, as compared to WT controls. In particular, melatonin was less effective in PPAR‐α KO, compared to WT mice, as evaluated by inhibition of the degree of spinal cord inflammation and tissue injury, neutrophil infiltration, pro‐inflammatory cytokine expression, NF‐κB activation, inducible nitric‐oxide synthase (iNOS) expression. This study indicates that PPAR‐a can contribute to the anti‐inflammatory activity of melatonin in SCI.
Astrocyte mitochondrial trifunctional protein mediates the neuroprotective effect of thyroid hormone after stroke in mice
Authors: Sifuentes Mikaela
Published: November 2023

Stroke is a leading cause of death and disability in the United States, however few treatments options exist for patients. Thyroid hormone is observed to reduce stroke lesion size in mice, but the mechanism for this neuroprotection is still unknown. Astrocytes are critical support cells in the brain that our lab has shown readily respond to thyroid hormone by increasing energy production through the mitochondrial trifunctional protein (MTP), a protein complex that facilitates fatty acid oxidation (FAO). As increased energy metabolism is protective against brain injury, we hypothesized that thyroid hormone reduces lesion size by stimulating astrocyte MTP. We observe that treatment with the active form of thyroid hormone (T3) stimulates an increase in ATP production in human astrocytes, which can be blocked by pretreatment with an irreversible FAO inhibitor. Astrocytes in the presence of T3 exhibit an increased survival under in vitro stroke conditions, while this effect is not seen in astrocytes lacking MTP. Pharmacological and genetic inhibition of MTP in vivo also reduces thyroid hormone reduction of lesion size, suggesting that MTP activity is a critical component in brain damage from focal ischemia. Finally, we demonstrate that thyroid hormone protection against ischemia is attenuated in the presence of low concentrations of fluoroacetate, an astrocyte‐specific mitochondrial inhibitor. Taken together, our research indicates that thyroid hormone reduces stroke damage by stimulating astrocyte metabolism in an MTP‐dependent manner.

Support or Funding Information

Supported in part by the William & Ella Owens Medical Research Foundation.
GPCRs expressed by cancer‐associated fibroblasts are potential therapeutic targets in pancreatic cancer
Authors: Zhou Shu , Chang Sarah , McCann Thalia , Nishihara Hiroshi , French Randall , Lowy Andrew , Insel Paul
Published: November 2023

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stromal matrix (desmoplasia) composed of activated fibroblasts (PFs) /stellate cells (PSCs), immune/ inflammatory cells and other cell types. Considerable evidence implicates this unique tumor microenvironment, and in particular its stromal cells, as a key mediator of PDAC progression and drug resistance. The tumor stroma may thus be a target for the treatment of PDAC. Pancreatic cancer‐associated fibroblasts (CAFs), myofibroblast‐like cells, thought to be derived from PSCs and PFs, produce extracellular matrix proteins and, are responsible for the desmoplasia in PDAC. Blocking the activity of CAFs may thus be a means to improve the therapy and prognosis of PDAC. We hypothesized that G protein‐coupled receptors (GPCRs) expressed by pancreatic CAFs may be potential therapeutic targets for PDAC. As an initial effort to test this hypothesis, we used an unbiased approach, Taqman GPCR arrays that can assess ~360 non‐chemosensory GPCRs, as a means to identify and quantify the GPCRs expressed by pancreatic CAFs. Using CAFs isolated from the primary tumors of 5 patients, we discovered that CAFs share in their expression of 104 GPCRs, 35 of which had at least a 2‐fold increase in expression in CAFs compared to PSCs and PFs. An orphan GPCR, Orphan 1, is one of the most highly up‐regulated GPCRs in CAFs. We found that: 1) co‐culture of PSCs with a PDAC cell line (ASPC‐1) increases expression of fibrotic markers and Orphan 1 GPCR; 2) Transfection of Orphan 1 GPCR in PSCs increases the expression of fibrotic markers; 3) siRNA knockdown of Orphan 1 in CAFs decreases fibrotic marker expression and cytokine secretion. We conclude that a GPCRomic strategy can identify GPCRs that regulate pancreatic CAFs. One such GPCR, Orphan 1, contributes to pro‐fibrotic activities in CAFs and is a potential therapeutic target to blunt the fibrosis associated with pancreatic cancer.

Support or Funding Information

NIH 5T32CA121938 and R21 CA189477
The GPCR CXCR4 and Tyrosine Phosphatase SHP2 Interact and Signal via a Novel Consensus Motif
Authors: Belcastro Lili T. , Paulukinas Ryan D. , Jancina Anastasia , Moore Catherine C.
Published: November 2023

CXCR4, a chemokine G protein‐coupled receptor (GPCR), is essential for migration of neuronal and hematopoietic cells, as well as breast cancer cells during metastasis whereby CXCR4 dysregulation promotes a migratory and invasive phenotype. Normally following activation, CXCR4 is rapidly phosphorylated on Ser/Thr residues by GRKs, a key step in initiating adaptor recruitment, which promotes receptor desensitization and receptor trafficking to endocytic sites. Previously we identified that CXCR4 Ser/Thr phosphorylation is delayed upon SDF gradient sensing, and correlates with sustained signaling to a novel CXCR4‐SHP2‐ERK pathway. SHP2 is a protein tyrosine phosphatase (PTP) implicated in HER2(+) and triple‐negative breast cancers, whereby SHP2 transduces both mitogenic and migratory signals, driving hyperproliferation and invasion. SHP2 is also known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine‐based inhibitory consensus motifs), a hallmark found in inhibitory immune receptors with little to no evidence for its presence and function in GPCRs. We have identified a putative ITIM motif in CXCR4 that regulates SHP2 binding and signaling. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated within the putative ITIM motif, using a rSHP2 phosphatase assay, pAb development, and mutagenesis, 2) if SHP2 interaction with CXCR4 is disrupted upon ITIM Tyr mutation, by co‐immunoprecipitation, 3) if CXCR4 signaling to SHP2 is disrupted upon ITIM Tyr mutation, and 4) if differences in signaling are due to improper localization and/or function of the mutant, by ELISA. Our results demonstrate that rSHP2 phosphatase is active on CXCR4, suggesting pTyr of CXCR4. Specific pTyr antibodies are under development to confirm these results. Additionally, Tyr mutation within the ITIM alters CXCR4 phosphorylation, suggesting pTyr of CXCR4 occurs within this motif. Furthermore, Tyr mutation disrupts CXCR4 immunocomplex formation with SHP2. Finally, sustained signaling to the SHP2‐ERK pathway is attenuated upon tyrosine mutation within the ITIM motif, and this effect is not due to improper receptor localization or function as demonstrated by cell surface expression and agonist‐dependent internalization. To date, our data support a working model that CXCR4 contains a functional ITIM motif capable of binding SHP2 and transducing sustained signaling to SHP2 upon SDF gradient sensing. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2.

Support or Funding Information

Supported by NIH GM‐097718 and PDH SAP4100057688 grants.
RNA‐seq based transcriptome profiling of human and mouse dorsal root ganglion reveals a potential role for Protease Activated Receptor 3 (PAR3) in pain processing
Authors: Price Theodore , Torck Andrew , Ray Pradipta , Hassler Shayne , Hoffman Justin , Boitano Scott , Dussor Gregory , Vagner Josef
Published: November 2023

The dorsal root ganglion (DRG) contains sensory neurons that innervate the surface of the body and many visceral organs. Included amongst these neurons are nociceptors, specialized neurons that detect damaging or potentially damaging stimuli, which are required for the detection of acute pain and play a key role in the development and maintenance of chronic pain states. RNA‐seq has recently been used to elucidate the transcriptome of this tissue in mouse and rat but the transcriptome of human DRG has not been explored. We obtained fresh, lumbar DRG tissue from female human donors and performed 75bp paired‐end polyA+ RNA‐sequencing on the Illumina platform. The sequenced fragments were mapped to the Gencode v14 reference transcriptome / hg19 reference genome to yield 80M mapped fragments, and relative transcript abundance in FPKM (Fragments Per Kilobase per Million mapped fragments) was quantified using the Tophat‐Cufflinks toolkit. We compared our RNA‐seq dataset to publicly available mouse DRG RNA‐seq data and performed integrative analysis with RNA‐seq data from several tissues associated with drug side effects (e.g. heart, small intestine, whole brain) to perform an unbiased search for conserved gene expression in DRG across both species. We find that there is broad conservation of known DRG and/or nociceptor enriched genes (e.g. P2XR3, SCN10A, SCN11A, NTRK1, MRGPRD) across mouse and human DRGs. Information theory approaches were used to identify tissue‐specific genes in human and mouse DRG compared to tissues 13 other tissues in human and mouse. We find strong correlation of expression across tissues between species for a few hundred DRG‐enriched transcripts, including known genes enriched in the DRG and previously unidentified ones. A conspicuous DRG enriched gene is F2RL2 which encodes PAR3. PAR3 expression in DRG is amongst the highest for all G protein coupled receptors (GPCRs) in human and mouse and mapping to existing cellular expression databases suggests neuronal expression in a population of nociceptors. To test the potential role of PAR3 in pain, we developed a novel ligand for this receptor using our synthetic tethered ligand discovery platform for PARs and show that it robustly activates calcium signaling in trigeminal ganglion neurons and causes mechanical hypersensitivity after hindpaw injection in mice but is devoid of activity at PAR2. Ongoing experiments are further evaluating the specificity of this compound. Our unbiased, human transcriptome approach to target discovery reveals PAR3 as a novel pain target.

Support or Funding Information

NIH grants NS073664 and NS065926
The Anti‐viral Agent Tenofovir Induces Oxidative Stress and Apoptosis in HK‐2 Cells
Authors: Murphy Rachel , Stafford Reagan , Petrasovits Brooke , Boone Megan , Valentovic Monica
Published: November 2023

Tenofovir disproxil fumarate (Viread) is a highly effective HIV antiviral drug approved for treating infections with Human Immunodeficiency Virus (HIV) and Hepatitis B. It is one of the first line drugs used to treat HIV and is efficacious in both novel and treatment‐experienced HIV patients. Tenofovir is administered orally as the prodrug tenofovir disoproxil fumarate (TDF), which is deesterified to the active drug tenofovir. However, renal damage is a major adverse effect associated with its use. Tenofovir can induce decreased glomerular filtration rate (GFR), renal failure, and Fanconi Syndrome. The exact mechanism of this toxicity remains unknown, largely due to limited experimental models. Our laboratory has established that clinically relevant concentrations of tenofovir are toxic within 72 h. The purpose of this study was to investigate the cellular mechanism of cytotoxicity in a human renal proximal tubular epithelial cell line (HK‐2). Tenofovir (TFV) is the active form and was used for all studies. HK‐2 cells were seeded and grown to confluency for 48 h followed by a 72 h exposure to 0–30 uM tenofovir. The vehicle was phosphate buffered saline (PBS). Cell viability was assessed using the MTT assay. Tenofovir induced a loss of cell viability when compared to vehicle within 48–72h but did not alter MTT following a 24 h exposure. Mitochondrial dysfunction was assessed by measuring ATP and ADP levels. A 72 h exposure to tenofovir drastically reduced (p<0.05) ATP levels compared to vehicle control. Oxidative stress was assessed using OxyBlot and showed an increase in protein carbonylation after 72 h exposure to 5–30 uM tenofovir. Oxidative stress induced by tenofovir was sufficient to induce an increase in 4‐hydroxynonenal (4‐HNE) adducted proteins and 3‐nitrotyrosine (3‐NT) addition to proteins. Tenofovir also activated cleavage of Caspase 3 and 9 relative to vehicle. These studies suggest that mitochondrial stress and apoptosis occur in HK‐2 cells treated with tenofovir.

Support or Funding Information

Supported by NASA West Virginia Space Grant Consortium Graduate Fellowship to Rachel Murphy.
Fenofibrate upregulates SHP expression but does not alter CYP2D6 expression
Authors: Kent Rebecca , Jeong Hyunyoung
Published: November 2023

Cytochrome P450 (CYP) 2D6 is a major drug‐metabolizing enzyme, responsible for eliminating 25% of marketed drugs. Factors involved in transcriptional regulation of CYP2D6 are poorly understood, and we recently identified SHP as a negative regulator of CYP2D6 expression (1). Factors that alter SHP expression could influence CYP2D6 expression, and therefore the extent of CYP2D6‐mediated drug metabolism. Fenofibrate has been previously reported to upregulate SHP expression in mouse liver (2). Our objective was to determine whether fenofibrate negativley alters CYP2D6 expression via upregulating SHP expression. CYP2D6‐humanized transgenic mice were administered with fenofibrate (100 mg/kg/day intraperitoneally for 5 days) or vehicle control. Hepatic mRNA and protein expression levels of CYP2D6 and SHP were measured. Results showed that while mRNA levels of SHP did not differ between the groups, protein levels of SHP increased by 2‐fold in fenofibrate‐treated mice. Despite the increased SHP protein levels, CYP2D6 expression did not decrease at the mRNA or protein levels. Similar results were observed in human hepatocytes treated with fenofibrate (at 200 μM for 48 hr). These results indicate that has minimal effects on CYP2D6 expression although it enhances SHP expression.

Support or Funding Information

This study was supported by NIH (GM112746 and HD065532).
Identification of Novel Probiotics to Modify Appetite and Satiety Directly Targeting the Ghrelin Receptor
Authors: Fuentes Cristine Torres , Schellekens Harriët , Hoevenaars Naomi , Ross Paul , Roy Bernard , Stanton Catherine , Dinan Timothy G. , Cryan John F.
Published: November 2023

Ghrelin is a stomach‐derived peptide that acts as orexigenic hormone in the hypothalamus stimulating food intake through the growth hormone secretagogue receptor (GHS‐R1a). Dysregulated ghrelin signalling may contribute to the development of metabolic disorders such as obesity. Current pharmacologic anti‐obesity treatments lack efficacy and have shown severe side effects, highlighting the urgent need for novel strategies contributing to the maintenance of a healthy weight.

Natural products are receiving special consideration as sources of bioactives with beneficial health effects which may be safer for consumers than synthetic therapeutics. One of the most investigated natural bioactives are live microorganisms with health benefit effects, also known as probiotics. Thus, food products containing probiotics are receiving increased scientific and commercial focus in recent years. These probiotic‐mediated beneficial effects on human health are due to changes in the gut microbiota via oral delivery of viable strains of bacteria that are then integrated into the gut system. Gut microbiota ferment nutrients and secrete bioactive compounds inducing several responses within the intestinal mucosa and controlling the gut's barrier and endocrine functions.

In this study we investigate the ability of different probiotic bacteria to modulate the GHS‐R1a receptor. Bacteria were cultured overnight under anaerobic conditions at 37°C. Culture supernatants were collected by centrifugation, resuspended in 1× Hanks balanced salt solution containing 20mM HEPES, and incubated in anaerobiosis for 4 h. Afterwards, supernatants were used to investigate the probiotics ghrelin receptor activation potential by calcium mobilization and receptor internalization assays in human embryonic kidney cells (Hek) stably expressing the GHS‐R1a receptor.

Different Lactobacillus and Bifidobacterium strains with ability to modulate the ghrelin receptor have been identified. Future investigations, including identification of the bioactives present in the probiotic supernatants as well as in vivo studies supporting these findings, are needed in order to confirm their potential as anti‐obesity therapeutics.

Support or Funding Information

Work funded by CREMO SA
Disruption of Gravin's Scaffolding Protects against Isoproterenol Induced Heart Failure
Authors: McConnell Bradley , Suryavanshi Santosh , Fa'ak Faisal , Diaz Andrea Diaz , Singh Sonal
Published: November 2023

β‐adrenergic receptor (β‐AR) signaling is the primary mechanism to increase cardiac contractility. However, chronic β‐AR stimulation, which occurs in HF, results in impairment of the β‐adrenergic pathway, leading to reduced contractility due in part by desensitization of these receptors. To induce contractility, β‐ARs signal through protein kinase A (PKA)‐dependent phosphorylation, which affects calcium homeostasis. PKA binds to A‐kinase anchoring proteins (AKAPs), which localize PKA and its substrates to defined intracellular locations. Gravin, an AKAP that targets PKA, protein kinase C (PKC) and protein phosphatase 2B (PP2B) to β2‐ARs, is involved in the modulation of the desensitization / resensitization cycle of β2‐ARs by facilitating PKA‐dependent phosphorylation and scaffolding of the β2‐AR complex. Thus, disruption of gravin's scaffolding action might prove beneficial to restore cardiac contractility during heart failure. In our studies, we used isoproterenol to chronically stimulate the β‐ARs to induce heart failure. We found that gravin‐t/t mice, harboring the truncated non‐functional gravin, leads to restriction of receptor desensitization during chronic β‐AR stimulation, due to loss in the assembly of the desensitization complex mediated by gravin's scaffolding. The β‐AR density is significantly higher in gravin‐t/t mice than wild‐type animals after 2 weeks of isoproterenol treatment. This decrease in the desensitization leads to the availability of more receptors on the heart to mediate chronotropy, ionotropy and lusitropy. Together, gravin‐t/t mice exhibited enhanced cardiac function in the presence of chronic β‐AR stimulation. PKA‐dependent substrates including cardiac troponin I (cTnI), phospholamban (PLB), and cardiac myosin binding protein C (cMyBPC), which show aberration activation patterns during heart failure, had normal phosphorylation levels in the gravin‐t/t mice, compared to the wild‐type mice. Gravin‐t/t mice also displayed enhanced myofilaments sensitivity to calcium compared to the wild‐type mice. Our results indicate that gravin plays an important role in regulating the β‐ AR pathway and disruption of its scaffolding augments cardiac function. Thus, blocking of gravin's scaffolding can be further explored as a potential therapeutic target for heart failure.

Support or Funding Information

Research reported in this poster was supported by the NHLBI institute of NIH under Award Number R01HL085487 (to B.K.M.) and R15HL124458 (to B.K.M.). This project was also supported by the Mouse Phenotyping Core at Baylor College of Medicine with funding from the NIH (U54 HG006348).
Pericyte Recruitment in a Diabetic Mouse Model of Corneal Neovascularization
Authors: Tavakol Daniel Naveed , Kelly‐Goss Molly , Yates Paul , Peirce‐Cottler Shayn
Published: November 2023

Angiogenesis, the generation of new blood vessels from a previously existing vasculature, is reinforced by the dynamic migration and signaling of wrapping support cells, termed pericytes, covering the capillary endothelium. Understanding pericyte‐endothelial cell interactions involved in angiogenesis is essential for developing therapies targeted at improving blood vessel growth and stabilization during disease, such as in diabetic wound healing. Previous work has established a connection between diminished pericyte coverage and delayed angiogenesis in diabetic wound healing models. To date, the location and identity of pericytes in healthy versus diabetic angiogenic networks has yet to be explored. Therefore, the objective of this study is to compare the extent of pericyte coverage in angiogenic networks of diabetic versus healthy mice.

Adult female heterozygous Ins2(Akita) diabetic mice and wild type mice (n=6 per group) underwent a silver nitrate chemical burn on the surface of the eye to induce corneal angiogenesis. Angiogenic sprouting into the previously avascular tissue was imaged daily using bright field microscopy. Three and seven‐days after injury, corneas were harvested and stained with CD31 (endothelial cell marker), NG2 (pericyte marker), and myosin heavy‐chain 11 (Myh11, pericyte and smooth muscle cell marker).

Using whole‐mount confocal imaging, Myh11+, NG2+ perivascular coverage over CD31+ endothelial cells was quantified in the vascular areas of the mouse models. There was a negative correlation between overlapping coverage of the two pericyte markers and glucose concentration (mg/dL) at days three and seven post‐stimulation (r‐square= 0.888 and 0.893, respectively). Subconjuctival hemorrhages per tissue in diabetic models were increased by 3.64‐fold and 4.00‐fold after three and seven days between glucose level and number of hemorrhages (r‐square= 0.787 and 0.831, respectively).

Diabetic neovessels in this injury model have diminished pericyte coverage relative to healthy mice, which is consistent with the presence of abundant subconjuctival hemorrhages in diabetic corneas. The comparison between data collected and glucose concentration in the blood is essential to understanding the relationship between these readings with the severity of diabetic effects, with an increase in glucose concentration equating to a decrease in pericyte‐supported vasculature. Together, these data suggest that reduced pericyte coverage on neovessels may contribute to the impaired angiogenesis that occurs during diabetic wound healing.

Support or Funding Information

University of Virginia Vice President for Research, Harrison Foundation, and the Arnold and Mabel Beckman Foundation
Deficiency for Smooth Muscle‐specific Insulin‐like Growth Factor I (IGF‐1) Receptor Increases Atherosclerotic Plaque Size and Promotes Plaque Destabilization
Authors: Sukhanov Sergiy , Higashi Yusuke , Snarski Patricia , Delafontaine Patrick
Published: November 2023

We have previously shown that systemic insulin‐like growth factor I (IGF‐1) infusion reduces atherosclerosis in Apoe‐null mice. We generated smooth muscle cells (SMC)‐specific IGF‐1 receptor (IGF‐1R)/Apoe‐double‐null mice (22KI) by crossing floxed IGF‐1R mice (FIR, control) with SM22a‐promoter‐driven Cre mice to study role of SMC IGF‐1R in atherogenesis. 22KI mice had >80% reduction in aortic IGF‐1R levels, 15±3% decrease in body weight (P<0.05) and 23±3% reduction in size of individual aortic SMC (P<0.05) suggesting that marked growth retardation seen in 22KI mice was mediated by SMC hypotrophy. 22KI mice had increase in aortic atherosclerotic plaque surface area (en face analysis, 22KI: 26±5% vs. FIR: 7±4%, P<0.001) and plaque cross‐sectional area (H&E staining, 22KI: 39±5% vs. FIR: 24±3%, P<0.05). Atherosclerotic plaque in 22KI mice had significantly reduced SMC levels (aSMA IHC: 70±3% decrease; calponin IHC: 76±3% decrease vs FIR) and increased SMC apoptosis (TUNEL assay: 2.5‐fold increase, P<0.01). 22KI's plaque had marked reduction in collagen levels (Trichrome staining, 22KI: 7±2% vs. FIR: 31±4%, P<0.05) and 4‐fold decrease in levels of collagen RNA‐binding protein LARP6. Aortic SMC isolated from 22KI mice had reduced proliferation (Edu assay), suppressed cell migration (wound healing assay) and increased sensitivity to oxidant‐induced apoptosis (TUNEL assay) and these effects were associated with inhibition of IGF‐1‐induced PI3 kinase/Akt‐dependent signaling.

We quantified features of former plaque disruption in brachiocephalic artery from 22KI and FIR mice. We found that 22KI's plaque has increased number of fibrous cap breaks; elevated intraplaque hemorrhage and increased fibrin deposition suggesting reduced plaque stability. In summary, SMC‐specific IGF‐1R deficiency increased atherosclerosis and reduced atherosclerotic plaque stability. Suppression of IGF‐1‐dependent and Akt‐mediated signaling may mediate pro‐atherogenic SMC phenotype in 22KI mice. These data established importance of SMC IGF‐1 signaling for atheroprotection.

Support or Funding Information

NIH/NHLBI
Bacterial Metabolite Indole Alters Inflammatory Signaling Cascades In Human Lymphatic Endothelial Cells
Authors: Muthuchamy Tarun , Lee Yang , Jayaraman Arul , Alaniz Robert C , Chakraborty Sanjukta
Published: November 2023

Pathological conditions, as metabolic syndrome (MetSyn) or obesity, are closely associated with inflammation‐induced alterations in the gut microbiota metabolites. Indole, a bacterial signal produced by Escherichia coli, has anti‐inflammatory effects on intestinal epithelial cells. As a key signaling molecule in the GI tract, indole has been found to have anti‐inflammatory effects on intestinal epithelial cells. Inflammatory conditions have been shown to directly alter the microbiota in the gut and several studies have linked these changes with onset of obesity and MetSyn, suggesting that the relative levels of indole could be changed in the intestine and mesentery under inflammatory conditions. We have previously shown that high fructose diet‐induced MetSyn in rats causes inflammatory conditions in the mesentery and impairs lymphatic contractile function. Hence, in this study we investigated whether the changes in the gut microbiota that occur under obesity, MetSyn or during other inflammatory conditions would influence lymphatic function. We hypothesize that indole reduces inflammation in lymphatic endothelial cells stimulated with dietary endotoxin, such as lipopolysaccharide (LPS). Human dermal lymphatic endothelial cells (HDLECs) were pretreated with a 100μM concentration of indole overnight, followed by LPS treatment for 24 hours. We used HDLECs treated with LPS or indole alone, and cells with no treatment were used as controls. Effects of indole on localization of NF‐κB and β‐catenin were determined by immunofluorescence. We also carried out quantitative RT‐PCR analysis to quantify the relative levels of expression of inflammatory marker genes, such as IL‐1b, c‐fos, c‐jun, MCP1, MIP1 and IL1A. Further, protein cytokine arrays were carried out to determine the relative levels of various inflammation associated cytokines and chemokines. Our immunofluorescence data indicate that indole decreases LPS‐mediated NF‐κB translocation to the nucleus. Further, the expression pattern of β‐catenin an important component of the adherens junction that controls endothelial cell permeability and normally present on the cell membrane was found to be diffused throughout the cytoplasm after LPS treatment. However, indole significantly decreased the LPS effects on β‐catenin and restored its expression on the cell membrane thereby tightening LEC junctions. Our quantitative RT‐PCR and protein cytokine data indicate that indole reduces the expression of many of the key proinflammatory chemokines, cytokines, and interleukins and thereby determines the balance between suppression of inflammation and progression of inflammatory signaling mechanisms. Thus, the data we have presented here provide the first evidence that a key signaling molecule of the gut microbiota, indole, reduces inflammation in the LECs, and has potential therapeutic value in the treatment of lymphatic inflammation associated pathological conditions.
Nrf2 deficiency exacerbates obesity‐induced neurovascular uncoupling in mice, mimicking the aging phenotype
Authors: Hertelendy Peter , Valcarcel‐Ares Marta Noa , Tarantini Stefano , Tucsek Zsuzsanna , Gautam Tripti , Farkas Eszter , Ungvari Zoltan , Csiszar Anna
Published: November 2023

Moment‐to‐moment adjustment of cerebral blood flow (CBF) via neurovascular coupling has a critical role in maintenance of healthy cognitive function. Previous studies demonstrate that obesity promotes cognitive impairment due to, at least in part, its deleterious effects on neurovascular coupling. Importantly, the effects of obesity are exacerbated in aging, but the underlying mechanisms remain elusive. There is increasing evidence that Nrf2/ARE signaling plays a critical role in vascular resilience to obesity and the metabolic syndrome and that aging is associated with progressive Nrf2 dysfunction, promoting microvascular oxidative stress. The present study was designed to test the hypothesis that Nrf2 deficiency contributes to neurovascular uncoupling and exacerbate cerebromicrovascular dysfunction induced by obesity. To test this hypothesis Nrf2+/+ and Nrf2−/− mice were fed a standard diet or an adipogenic high fat diet (HFD) and cortical neurovascular coupling responses were assessed. In control mice feeding HFD for 6 months significantly increased vascular oxidative stress and decreased endothelium‐mediated CBF responses (laser Doppler flowmetry) in the somatosensory cortex evoked by contralateral whisker stimulation. Nrf2 deficiency significantly exacerbated HFD‐induced neurovascular uncoupling, mimicking the aging phenotype. Together, these results provide additional evidence that intact Nrf2 signaling importantly contributes to cerebromicrovascular health, preserving neurovascular coupling mechanisms. Further studies are needed to elucidate the endothelial and astrocytic mechanisms impacted by Nrf2 deficiency and HFD‐induced obesity, determining the potential roles of oxidative stress and chronic low‐grade neuroinflammation.

Support or Funding Information

This work was supported by grants from the American Heart Association and the National Institute on Aging.
Western Diet Increases Inflammatory Responses and Atherosclerosis Progression via IGF‐1 Receptor in ApoE‐Deficient Mice
Authors: Wang Meifang , Higashi Yusuke , Wang Derek , Korthuis Ronald , Delafontaine Patrice
Published: November 2023

Atherosclerosis is the principal underlying cause of most cardiovascular disease‐related deaths, the leading cause of mortality in the US. Insulin‐like growth factor‐1 (IGF‐1) is the primary mediator of the effect of growth hormone on developmental growth which is expressed in vascular cells and monocytes/macrophages. But its role in atherosclerosis is unknown. Our previous studies show that lowered circulating IGF‐1 increases atherosclerosis progression in atherosclerosis‐prone apolipoprotein E‐deficient mice (ApoE−/−) while increased circulating IGF‐1 downregulates vascular pro‐inflammatory cytokine gene expression, reduces systemic and vascular oxidant stress, vascular cytokine expression, and decreases atherosclerosis progression. These findings led us to investigate whether the pro‐inflammatory phenotypes became more noticeable in ApoE−/− mice, in which IGF1R‐expression is deleted selectively in monocytes/macrophage (ApoE−/− LC), and whether consumption of a Western diet would change the interaction between monocytes and endothelium cells in these mice and IGF‐1R ApoE−/−‐flox mice (ApoE−/−‐FIR). To test these hypotheses, we compared baseline Monocyte Rolling (MR) and Monocyte Adhesion (MA) in both these mice fed with either normal mouse chow (NC) or Western diet (WD). There was no significant change in the pro‐inflammatory phenotype in mice with deficient IGF1R‐expression on macrophages in ApoE−/− mice. However, ingestion of a Western diet markedly increased MR and MA on ApoE−/− LC animals (ApoE−/− LC WD) compared to ApoE−/− LC NC mice, although there was no significant effect on ApoE−/−‐FIR mice. These findings indicate that the pro‐inflammatory phenotype that is enhanced by WD in ApoE−/− mice is further enhanced by specific IGF1R deficiency in monocytes/macrophages, and may consequently increase atherosclerosis progression.

Support or Funding Information

R01‐HL59976, P01‐HL ‐095486 & R01‐AA22108, RO1 HL070241 and HL 080682.
N‐acetylcysteine reverses established cardiac and renal fibrosis in a mouse model of heart failure
Authors: Rajapakse Niwanthi , Giam Beverly , Chu Po‐Yin , Kiriazis Helen , Du Xiao‐Jun , Kaye David M
Published: November 2023

Background

Patients with heart failure (HF) often develop secondary renal dysfunction and fibrosis plays a major pathological role in the development of both cardiac and renal dysfunction in these patients. We aimed to determine whether the antioxidant N‐acetylcysteine (NAC) can reverse cardiac and renal fibrosis in a transgenic mouse model of HF. These mice develop cardiomyopathy secondary to cardiac specific overexpression of mammalian sterile 20–like kinase 1 (MST‐1).1 Overexpression of MST‐1 in cardiomyocytes induces apoptosis1 which is relevant to human HF and we have previously demonstrated that mice with cardiac specific overexpression of MST‐1 (MST‐1 mice) develop extensive cardiac fibrosis by 13 weeks of age.2 The aim of the current study was to determine whether NAC can reverse cardiac and renal fibrosis in the setting of HF.

Methods

Minipumps were implanted subcutaneously in 18 week old male wild type (WT; n=6–20) and MST‐1 mice (n=6–10) to administer NAC (40 mg/kg/day) or saline for a period of 8 weeks. At the end of this treatment period, cardiac remodelling and function was assessed via echocardiography. Fibrosis in the heart and kidney tissues was quantified using Masson Trichrome staining.

Results

Cardiac perivascular and interstitial fibrosis were 2‐ and 19‐fold greater, respectively, in saline treated MST‐1 mice compared to saline treated WT (P ≤ 0.01). In NAC treated MST‐1 mice, perivascular and interstitial fibrosis were 37% and 68% less, respectively, compared to those treated with saline (P ≤ 0.05). Fractional shortening was 61% less in saline treated MST‐1 mice compared to saline treated WT. Fractional shortening was not significantly different between NAC treated MST‐1 mice and those treated with saline. Heart weight:body weight ratio was 27% greater in saline treated MST‐1 mice compared to saline treated WT (P < 0.05). This ratio was not significantly different between NAC treated MST‐1 mice and saline treated MST‐1 mice.

Renal tubulointerstitial and glomerular fibrosis were 23‐ and 2‐fold greater, respectively, in saline treated MST‐1 mice compared to saline treated WT (P ≤ 0.01). Of interest, renal tubulointerstitial and glomerular fibrosis were 99% and 67% less, respectively, in NAC treated MST‐1 mice compared to those treated with saline (P ≤ 0.01).

Conclusions

These data indicate that NAC can reverse cardiac and renal fibrosis in the setting of HF potentially by reducing oxidative stress.

Support or Funding Information

This work was supported by a NHMRC (Australia) program grant.
Individual fluid therapy vs. standardized high volume fluid infusion during kidney transplantation – evaluation of oedema development, oxidative stress and inflammation levels, using a porcine model
Authors: Nielsen Lise Have
Published: November 2023

The objective of the study is to evaluate the current fluid therapy distributed to kidney transplant patients, as to avoid overhydration.

Background

Many kidney transplant patients are severely overhydrated after the operation, which can cause prolonged hospital stays, higher risk of infection and delayed graft function of the kidney. To avoid overhydrated patients, fluid strategies have been undergoing evaluation and validation, and it is now generally agreed that a restrictive fluid regiment is healthier. In this study we will try to access the consequences of overhydration using tissue markers of oxidative stress and inflammation in the kidney, as to quantify the risks of the above mentioned negative results.

Methods

Using a brain dead porcine model on day one, two kidneys from a single pig is taken out, stored at 4°C for 18 hours (in a cold storage solution; “Bridge of life”™/Custodiol)and then transplanted into two different pigs (after removal of the native kidneys).

After reperfusion, the pigs are surveilled for 10 hours, during which blood and urine samples are collected. At the end of the experiment, the kidneys are removed and tissue samples are collected.

The individual fluid therapy is 2 mL/kg/hour (with added bolus of 500 mL if the stroke volume changes more than 12% when challenged), the standardized high volume infusion is 10 mL/kg/hour, and both regimes are supplemented by bolus infusion if MAP should drop below 60 mmHg.

The recipient pigs are randomly divided into 4 groups: 1) Individual Goal Directed Fluid Therapy w/o noradrenaline infusion, 2) Individual Goal Directed Fluid Therapy with noradrenaline infusion, 3) Standardized High Volume Fluid Infusion w/o noradrenaline infusion and 4) Individual Goal Directed Fluid Therapy with noradrenaline infusion.

The blood, urine and tissue samples will be tested for markers of oxidative stress and inflammation; ex. glutathione, NGAL, COX‐2, TBARS.

Results/discussion

As the experiment is still ongoing, no results have been obtained yet.

However, every transplanted kidney has developed oedema after reperfusion, no matter what fluid treatment was given – statistics will prove whether or not the kidneys receiving standardized fluid therapy is developing more distinct oedema.

We hope to discuss the effect of the infusion of low dose noradrenaline and evaluate the exact dose recommended. We would also discuss whether or not it is the difference in fluid amount that makes a difference, or rather the distribution of the fluids.

Conclusion

The results are expected to influence the clinical protocol used to direct the treatment of kidney transplant patients, hereby eliminating overhydration as a delaying factor in the curative process.