The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Lipid Nanoparticles Deliver the Therapeutic VEGFA mRNA In Vitro and In Vivo and Transform Extracellular Vesicles for Their Functional Extensions.

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells' own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells' response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs.

Towards fully automated segmentation of rat cardiac MRI by leveraging deep learning frameworks.

Automated segmentation of human cardiac magnetic resonance datasets has been steadily improving during recent years. Similar applications would be highly useful to improve and speed up the studies of cardiac function in rodents in the preclinical context. However, the transfer of such segmentation methods to the preclinical research is compounded by the limited number of datasets and lower image resolution. In this paper we present a successful application of deep architectures 3D cardiac segmentation for rats in preclinical contexts which to our knowledge has not yet been reported. We developed segmentation models that expand on the standard U-Net architecture and evaluated models separately trained for systole and diastole phases (2MSA) and a single model trained for all phases (1MSA). Furthermore, we calibrated model outputs using a Gaussian process (GP)-based prior to improve phase selection. The resulting models approach human performance in terms of left ventricular segmentation quality and ejection fraction (EF) estimation in both 1MSA and 2MSA settings (Sørensen-Dice score 0.91 ± 0.072 and 0.93 ± 0.032, respectively). 2MSA achieved a mean absolute difference between estimated and reference EF of 3.5 ± 2.5%, while 1MSA resulted in 4.1 ± 3.0%. Applying GPs to 1MSA enabled automating systole and diastole phase selection. Both segmentation approaches (1MSA and 2MSA) were statistically equivalent. Combined with a proposed cardiac phase selection strategy, our work presents an important first step towards a fully automated segmentation pipeline in the context of rat cardiac analysis.

Functionalized lipid nanoparticles for subcutaneous administration of mRNA to achieve systemic exposures of a therapeutic protein.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.

Identification and Optimization of Pyrrolidine Derivatives as Highly Potent Ghrelin Receptor Full Agonists.

Muscle atrophy and cachexia are common comorbidities among patients suffering from cancer, chronic obstructive pulmonary disease, and several other chronic diseases. The peptide hormone ghrelin exerts pleiotropic effects including the stimulation of growth hormone secretion and subsequent increase of insulin-like growth factor-1 levels, an important mediator of muscle growth and repair. Ghrelin also acts on inflammation, appetite, and adipogenesis and therefore has been considered a promising therapeutic target for catabolic conditions. We previously reported on the synthesis and properties of an indane based series of ghrelin receptor full agonists which led to a sustained increase of insulin-like growth factor-1 in a dog pharmacodynamic study. Herein we report on the identification of a series of pyrrolidine or piperidine based full agonists and attempted optimization to give compounds with profiles suitable for progression as clinical candidates.

Pharmacological modulation of colorectal distension evoked potentials in conscious rats.

BACKGROUND: Cerebral evoked potentials (CEP) induced by colorectal distension (CRD) in conscious rats provides a novel method in studies of visceral sensitivity. The aim of this study was to explore the pharmacological effect on CEP of compounds known to reduce the visceromotor response to CRD. METHODS: Epidural electrodes were chronically implanted in eight female Sprague-Dawley rats. Evoked potentials were elicited by colorectal rapid balloon distensions (100 ms, 80 mmHg) and the effect of WIN55 (cannabinoid CB receptor agonist), clonidine (adrenergic α2 receptor agonist), MPEP (mGluR5 receptor antagonist), pregabalin (ligand of α2δ subunits in voltage-gated calcium channels) and baclofen (GABA-B receptor agonist) on amplitudes and latency of CEP were determined. RESULTS: WIN55 (0.1 µmol kg-1), clonidine (0.05 µmol kg-1), MPEP (10 µmol kg-1) and pregabalin (200 µmol kg-1) caused a significant, p < 0.05, reduction of the N2 to P2 peak-to-peak amplitude by 23 ±â€¯8%, 25 ±â€¯8%, 39 ±â€¯5%, and 47 ±â€¯6% respectively. Baclofen (9 µmol kg-1) induced a prolongation of the N2 peak latency of 18 ±â€¯4% but had no significant effect on the amplitudes. CONCLUSION: The obtained results suggest that MPEP, WIN55, clonidine, and pregabalin reduce visceral nociceptive input to the brain, whereas the lack of effect of baclofen on CRD evoked CEP amplitudes suggest that the effect on VMR is not due to a direct analgesic effect. Brain responses to colorectal distension provide a useful tool to evaluate pharmacological effects in rats and may serve as a valuable preclinical model for understanding pharmacological mechanisms related to visceral sensitivity.

Rheumatic Disease: Protease-Activated Receptor-2 in Synovial Joint Pathobiology.

Protease-activated receptor-2 (PAR2) is one member of a small family of transmembrane, G-protein-coupled receptors. These receptors are activated via cleavage of their N terminus by serine proteases (e.g., tryptase), unveiling an N terminus tethered ligand which binds to the second extracellular loop of the receptor. Increasing evidence has emerged identifying key pathophysiological roles for PAR2 in both rheumatoid arthritis (RA) and osteoarthritis (OA). Importantly, this includes both pro-inflammatory and destructive roles. For example, in murine models of RA, the associated synovitis, cartilage degradation, and subsequent bone erosion are all significantly reduced in the absence of PAR2. Similarly, in experimental models of OA, PAR2 disruption confers protection against cartilage degradation, subchondral bone osteosclerosis, and osteophyte formation. This review focuses on the role of PAR2 in rheumatic disease and its potential as an important therapeutic target for treating pain and joint degradation.

Identification and Pharmacological Profile of an Indane Based Series of Ghrelin Receptor Full Agonists.

Cachexia and muscle wasting are very common among patients suffering from cancer, chronic obstructive pulmonary disease, and other chronic diseases. Ghrelin stimulates growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGF-1 plasma levels. The activation of the GH/IGF-1 axis leads to an increase of muscle mass and functional capacity. Ghrelin further acts on inflammation, appetite, and adipogenesis and for this reason was considered an important target to address catabolic conditions. We report the synthesis and properties of an indane based series of ghrelin receptor full agonists; they have been shown to generate a sustained increase of IGF-1 levels in dog and have been thoroughly investigated with respect to their functional activity.

Effect of pancreatic polypeptide on gastric accommodation and gastric emptying in conscious rats.

Pancreatic polypeptide (PP) is an anorexigenic hormone released from pancreatic F cells upon food intake. We aimed to determine the effect of PP on gastric accommodation and gastric emptying in conscious Wistar HAN rats to investigate whether effects on motor function could contribute to its anorexigenic effects. Intragastric pressure (IGP) was measured through a chronically implanted gastric fistula during the infusion of a nutrient meal (Nutridrink; 0.5 ml/min). Rats were treated with PP (0, 33 and 100 pmol·kg(-1)·min(-1)) in combination with N(G)-nitro-L-arginine methyl ester (L-NAME; 180 mg·kg(-1)·h(-1)), atropine (3 mg·kg(-1)·h(-1)), or vehicle. Furthermore, the effect of PP was tested after subdiaphragmal vagotomy of the stomach. Gastric emptying of a noncaloric and a caloric meal after treatment with 100 pmol·kg(-1)·min(-1) PP or vehicle was compared using X-rays. PP significantly increased IGP during nutrient infusion compared with vehicle (P < 0.01). L-NAME and atropine significantly increased IGP during nutrient infusion compared with vehicle treatment (P < 0.005 and 0.01, respectively). The effect of PP on IGP during nutrient infusion was abolished in the presence of L-NAME and in the presence of atropine. In vagotomized rats, PP increased IGP compared with intact controls (P < 0.05). PP significantly delayed gastric emptying of both a noncaloric (P < 0.05) and a caloric (P < 0.005) meal. PP inhibits gastric accommodation and delays gastric emptying, probably through inhibition of nitric oxide release. These results indicate that, besides the well-known centrally mediated effects, PP might decrease food intake through peripheral mechanisms.

Translational aspects of rectal evoked potentials: a comparative study in rats and humans.

Inconsistencies between species has stunted the progress of developing new analgesics. To increase the success of translating results between species, improved comparable models are required. Twelve rats received rectal balloon distensions on 2 different days separated by 24.3 (SD 24.6) days. Rectal balloon distensions were also performed in 18 humans (mean age: 34 yr; range: 21-56 yr; 12 men) on two separate occasions, separated by 9.3 (SD 5.5) days. In rats, cerebral evoked potentials (CEPs) were recorded by use of implanted skull-electrodes to distension pressure of 80 mmHg. In humans surface electrodes and individualized pressure, corresponding to pain detection threshold, were used. Comparison of morphology was assessed by wavelet analysis. Within- and between-day reproducibility was assessed in terms of latencies, amplitudes, and frequency content. In rats CEPs showed triphasic morphology. No differences in latencies, amplitudes, and power distribution were seen within or between days (all P ≥ 0.5). Peak-to-peak amplitude between the first positive and negative potential were the most reproducible characteristic within and between days (evaluated by intraclass correlation coefficients, ICC) (ICC = 0.99 and ICC = 9.98, respectively). In humans CEPs showed a triphasic morphology. No differences in latencies, amplitudes, or power distribution were seen within or between days (all P ≥ 0.2). Latency to the second negative potential (ICC = 0.98) and the second positive potential (ICC = 0.95) was the most reproducible characteristic within and between days. A unique and reliable translational platform was established assessing visceral sensitivity in rats and humans, which may improve the translational process of developing new drugs targeting visceral pain.

Effect of muscarinic and nicotinic receptor antagonism on rat gastric motor activity.

BACKGROUND/AIMS: Our aim was to investigate whether muscarinic and nicotinic receptors mediate nitric oxide release during motor events in the rat stomach. METHODS: Isolated rat stomach volume changes were monitored in an organ bath setup with an intragastric balloon coupled to a barostat and studied in basal conditions and during electrical vagal stimulation (EVS). In conscious rats, the intragastric pressure (IGP) was measured during test meal infusion. RESULTS: In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME; 0.1 mmol/l), EVS induced significant gastric contractions (mean +/- SEM = 0.27 +/- 0.04 ml; n = 6) that could be blocked by atropine (3 micromol/l) and hexamethonium (0.1 mmol/l). In the presence of atropine and/or hexamethonium, EVS-induced relaxations could not be blocked by L-NAME, while exogenous nitric oxide could still relax the stomach. In conscious rats, atropine (1 mg kg(-1)) initially decreased IGP, while during further distension it increased IGP. In the presence of L-NAME (30 mg kg(-1)) atropine consistently decreased IGP. L-NAME alone significantly increased IGP during the test meal infusion, but this effect was reduced in the presence of atropine. CONCLUSION: These findings indicate a role for nicotinic and muscarinic receptors in the vagal-stimulation-induced activation of nitrergic nerves in the rat stomach.

A novel method for study of gastric mechanical functions in conscious mice.

A novel method has been developed for simultaneous study of gastric emptying, antral motility, and gastric muscle tone in conscious mice. Intragastric pressure was measured during infusion of an X-ray-opaque, viscous meal through a chronically implanted gastric fistula (0.25 ml/min). Compared with vehicle treatment, molsidomine (nitric oxide donor) and atropine (muscarinic receptor antagonist) treatment significantly reduced the area under the intragastric pressure curve (AUC) by 37 +/- 4% and 35 +/- 3%, respectively, (mean +/- S.E.M.) whereas N (G)-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase inhibitor) significantly increased the AUC by 20 +/- 3%. Atropine also significantly reduced the frequency and amplitude of stomach contraction-induced intragastric pressure waves while molsidomine only reduced the frequency. Gastric emptying, as assessed by X-ray imaging, was significantly delayed after L-NAME and atropine treatment. This methodology is the first to enable simultaneous assessment of gastric emptying, antral motility, and gastric tone in conscious mice and confirmed the important role of nitrergic and cholinergic innervation.

A novel method to assess gastric accommodation and peristaltic motility in conscious rats.

OBJECTIVE: To simultaneously study gastric accommodation and peristaltic motility in the whole stomach of conscious rats by measuring intragastric pressure (IGP) during test-meal infusion. MATERIAL AND METHODS: After an overnight fast, a test-meal infusion system and a catheter to measure IGP were connected to a chronically implanted gastric fistula. IGP was measured during infusion of an X-ray-opaque, non-nutritious viscous test meal (0.25-2 ml min(-1)); gastric motility and emptying were assessed by X-ray fluoroscopy. Peristaltic motility-induced IGP waves were quantified as a motility index (wave amplitude divided by wavelength). Experiments were performed in Sprague-Dawley (SD) rats and in the high-anxiety Wistar Kyoto (WKY) rats. Moreover, the effects of 30 mg kg(-1) NG-nitro-L-arginine methyl ester (L-NAME), 1 mg kg(-1) atropine or 20 mg kg(-1) molsidomine were tested in SD rats. RESULTS: Compared with SD rats, IGP increased significantly faster during stomach distension in WKY rats, indicating impaired accommodation in the latter strain. Motility indices did not differ between the two strains. L-NAME significantly increased IGP during stomach distension, indicating decreased gastric accommodation. However, no change in motility indices was observed with L-NAME. Treatment with atropine significantly increased IGP and decreased motility indices, indicating decreased gastric accommodation and motility. Molsidomine significantly decreased IGP during stomach distension but did not affect motility. The results correspond to X-ray observations, and confirm literature data. CONCLUSIONS: We conclude that IGP measurement during test-meal infusion represents an efficient and novel method to compare gastric accommodation and peristaltic motility in the whole stomach of conscious rats.

Predicting and monitoring colitis development in mice by micro-computed tomography.

BACKGROUND: Computed tomography (CT) has been developed as a tool for monitoring human inflammatory bowel disease (IBD). The aim of this study was to evaluate colon wall thickness as a noninvasive marker in the dextran sodium sulfate (DSS) mouse model of colitis using micro-CT. METHODS: Mice were examined by micro-CT 1, 2, or 4 times between day 0 (d0) and d26 after induction of colitis to document the kinetics of changes in colon wall thickness and its relation to colitis development. RESULTS: DSS-treated mice displayed a significantly thicker colon wall at all timepoints (days 5, 8, 12, 19, and 26) investigated compared to healthy controls. Colon wall thickness showed a good correlation to the macroscopic grading of colitis (r = 0.81). The increase in colon wall thickness occurred mainly during the acute phase of colitis (between days 5 and 12) and did not progress much further in the chronic phase of colitis (d26). Colon wall thickness at d26 was thereby predicted by measurements at d12. All mice did not respond equally to DSS and this difference was manifest during the first 2 weeks of colitis, providing an important tool in stratifying responders from nonresponders. CONCLUSIONS: While the potential impact of handling and anesthesia should be considered on repeated micro-CT, irradiation exposure during repeated micro-CT did not affect the development of colitis. Thus, the results suggest that micro-CT can be used for monitoring and prediction of the inflammatory response in mouse colitis in future therapeutic studies.

Early identification of drug-induced impairment of gastric emptying through physiologically based pharmacokinetic (PBPK) simulation of plasma concentration-time profiles in rat.

Inhibition of gastric emptying rate can have adverse effects on the absorption of food and nutrients. The absorption phase of the plasma concentration-time profile of a compound administered orally to pre-clinical species reflects among others, the gastric and intestinal transit kinetics, and can thus assist in the early identification of delayed gastric emptying. The purpose of this article is to demonstrate the value of Physiologically Based Pharmacokinetic (PBPK) modelling in the early identification of drug induced impairment of gastric emptying from pharmacokinetic profiles. To our knowledge, this is first time that the value of a generic PBPK model for hypothesis testing has been demonstrated with examples. A PBPK model built in-house using MATLAB package and incorporating absorption, metabolism, distribution, biliary and renal elimination models has been employed for the simulation of concentration-time profiles. PBPK simulations of a few compounds that are currently in drug discovery projects show that the observed initial absorption phase of their concentration-time profiles in rat were consistent with reduced gastric emptying rates. The slow uptake of these compounds into the systemic circulation is reflected in their pharmacokinetic profiles but it is not obvious until PBPK simulations are done. Delayed gastric emptying rates of these compounds in rats were also independently observed in x-ray imaging. PBPK simulations can provide early alerts to drug discovery projects, besides aiding the understanding of complex mechanisms that determine the lineshapes of pharmacokinetic profiles. The application of PBPK simulations in the early detection of gastric emptying problems with existing data and without the need to resort to additional animal studies, is appealing both from an economic and ethical standpoint.

Measurement of MR signal and T2* in lung to characterize a tight skin mouse model of emphysema using single-point imaging.

PURPOSE: To evaluate whether MRI signal and T2* measurements of lung tissue acquired at ultrashort detection times (tds) can detect emphysematous changes in lungs. MATERIALS AND METHODS: MR signal intensity of in vivo mouse lungs was measured at 4.7 T at tds of 0.2 and 0.4 msec using single-point imaging (SPI). T2* was calculated from the measurements obtained at the two tds. Two groups of 8- and 30-week-old Tight Skin (TS) and aged-matched CB57BL/6 mice were examined. The TS mice spontaneously developed emphysema-like alveolar enlargement. In vivo micro-computed tomography (microCT) scanning and histology were used as reference methods. RESULTS: MR signal and T2* were significantly lower in the lungs of TS mice than in controls. There were no significant differences between the different age groups. MR signal in lung parenchyma correlated linearly (P < 0.0001, r = 0.89) with microCT mass density, and T2* correlated linearly (P < 0.0001, r = -0.91) with the alveoli size (mean linear intercept [MLI]). CONCLUSION: The MR signal intensity and T2* measured at short tds can be used as imaging biomarkers to characterize parenchyma density and alveolar size, respectively.

Effective blood signal suppression using double inversion-recovery and slice reordering for multislice fast spin-echo MRI and its application in simultaneous proton density and T2 weighted imaging.

PURPOSE: To design a multislice double inversion-recovery fast spin-echo (FSE) sequence, with k-space reordered by inversion time at slice position (KRISP) technique, to produce black-blood vessel wall magnetic resonance imaging (MRI). MATERIALS AND METHODS: In this sequence, central k-space sampling for each slice is required at inversion time (TI) of the blood signal. To fill the entire k-space, the peripheral lines are obtained less or greater the TI and using a rotating slice order. Blood flow signal suppression was first evaluated using a phantom. Simulation studies were used to investigate FSE image quality. The final sequence was then applied to the rabbit abdominal aorta MRI at 4.7 T. RESULTS: In the flow phantom study, artifacts from slow-flowing water were substantially reduced by the KRISP technique; residual water spins were dephased by the strong phase-encoding gradient required for peripheral k-space. These dephased spins flowed into the slice plane where the center of k-space was being acquired at the TI of the flowing water signal. Multislice black-blood MR images were successfully obtained in the rabbit abdomen using the sequence with the k-trajectory optimized by the simulation study. CONCLUSION: The KRISP technique was effective both in multislice double inversion-recovery FSE and in blood signal suppression.

Reduced frontotemporal perfusion in psychopathic personality.

Several brain-imaging studies have found associations between aberrant functioning in the frontal and temporal lobes and violent offending. We have previously reported decreased frontotemporal perfusion unrelated to psychosis, substance abuse, or current medication in 21 violent offenders. In the present study, we compared the regional cerebral blood flow (rCBF) in a new group of 32 violent offenders to scores on the Psychopathy Checklist-Revised (PCL-R), which rates two aspects of psychopathy: disturbed interpersonal attitudes (Factor 1) and impulsive antisocial behavior (Factor 2). A recently proposed model has split Factor 1 into a new Factor 1 (deceitful interpersonal style), a new Factor 2 (affective unresponsiveness), and a Factor 3, which approximately corresponds to the old Factor 2. The rCBF was assessed by single-photon emission computed tomography (SPECT) with technetium-99m-d,l-hexamethylpropyleneamine oxime (HMPAO) in regions of interest (ROIs) placed in accordance with fusioned magnetic resonance images (MRI) and SPECT scans. Significant negative correlations were found between interpersonal features of psychopathy (the old and especially the new Factor 1) and the frontal and temporal perfusion. The two most clearly associated ROIs were the head of the caudate nuclei and the hippocampi. These findings in a group of violent offenders living under the same conditions, which reduced the number of state-related confounders, add to the evidence indicating that aberrant frontotemporal activity may be a factor in violent behavior.