CXCL12/CXCR4 pathway as a novel therapeutic target for RNF213-associated pulmonary arterial hypertension.

Genetic backgrounds of patients with pulmonary arterial hypertension (PAH) were not fully investigated. A variant of c.14429G > A (p.Arg4810Lys) in the ring finger protein 213 gene (RNF213) was recently identified as a risk allele for poor treatment response and poor clinical prognosis in patients with PAH. However, the molecular mechanisms of the RNF213 p.Arg4810Lys variant in development of PAH are unknown. We investigated the underlying molecular mechanisms of RNF213-associated vasculopathy using an in vivo mouse model. RNF213+/p.Arg4828Lys mice, harboring the heterozygous RNF213 p.Arg4828Lys variant corresponding to the p.Arg4810Lys variant in humans, were created using the CRISPR-Cas9 system to recapitulate the genetic status of PAH patients. RNF213+/p.Arg4828Lys mice had a significant elevation of the right ventricular systolic pressure, hypertrophy of the right ventricle, and increased thickness of the pulmonary arterial medial wall compared with wild-type mice after 3 months of exposure to a hypoxic environment. C-X-C motif chemokine ligand 12 (CXCL12), a C-X-C chemokine receptor type 4 (CXCR4) ligand, was significantly elevated in the lungs of RNF213+/p.Arg4828Lys mice, and PAH was ameliorated by the administration of a CXCR4 antagonist. CXCL12-CXCR4 is an angiogenic chemokine axis, and immunohistochemistry demonstrated an increase in CXCR4 in vimentin-positive spindle-shaped cells in adventitia and interstitial lesions in RNF213+/p.Arg4828Lys mice and lung specimens from severe PAH patients with the RNF213 p.Arg4810Lys variant. We confirmed a cause-and-effect relationship between the RNF213 p.Arg4810Lys variant and PAH via the CXCL12-CXCR4 pathway. The findings in this study suggest that targeting this pathway might be a novel therapeutic strategy for RNF213-associated vasculopathy.

Impact of daily administration of blackberry extract on gerbil model of transient cerebral ischemia.

PURPOSE: Blackberries are rich in polyphenols and are a human health food continuously consumed to improve health and reduce diseases caused by aging. Herein, we evaluated the effects of daily blackberry administration before and after transient cerebral ischemia in gerbils. METHODS: Blackberry extract (BBE) was orally administered twice a day for two weeks to protect against ischemic events during continuous administration. On the seventh day after administration, the bilateral common carotid arteries were transiently occluded for 5 min. To verify its therapeutic effect, BBE was administered after ischemia using a similar protocol without pre-administration. In both experiments, the number of viable neurons in the CA1 region of the hippocampus was assessed seven days after ischemic treatment. RESULTS: The number of neurons in the group treated with BBE before ischemia was higher than that in the group treated with distilled water (p = 0.0601), and similar to that in the control group. In the BBE administration experiments after ischemia, the number of neurons was significantly reduced compared to that in the control group (p < 0.0001). CONCLUSIONS: Continuous BBE intake is expected to prevent or ameliorate ischemic events such as transient cerebral ischemia.

Potential of Enzymatically Synthesized Hemozoin Analog as Th1 Cell Adjuvant.

Hemozoin (Hz) is a heme crystal produced during malaria infection that stimulates immune cells, leading to the production of cytokines and chemokines. The immunostimulatory action of Hz has previously been applied in the development of alternative adjuvants. Crystallization of hemin is a chemical approach for producing Hz. Here, we focused on an enzymatic production method for Hz using the heme detoxification protein (HDP), which catalyzes heme dimer formation from hemin in Plasmodium. We examined the immunostimulatory effects of an enzymatically synthesized analog of Hz (esHz) produced by recombinant Plasmodium falciparum HDP. Enzymatically synthesized Hz stimulates a macrophage cell line and human peripheral mononuclear cells, leading to the production of interleukin (IL)-6 and IL-12p40. In mice, subcutaneous administration of esHz together with an antigen, ovalbumin (OVA), increased the OVA-specific immunoglobulin (Ig) G2c isotype level in the serum, whereas OVA-specific IgG1 was not induced. Our findings suggest that esHz is a useful Th-1 cell adjuvant.

A neural cell automated analysis system based on pathological specimens in a gerbil brain ischemia model.

PURPOSE: Amid rising health awareness, natural products which has milder effects than medical drugs are becoming popular. However, only few systems can quantitatively assess their impact on living organisms. Therefore, we developed a deep-learning system to automate the counting of cells in a gerbil model, aiming to assess a natural product's effectiveness against ischemia. METHODS: The image acquired from paraffin blocks containing gerbil brains was analyzed by a deep-learning model (fine-tuned Detectron2). RESULTS: The counting system achieved a 79%-positive predictive value and 85%-sensitivity when visual judgment by an expert was used as ground truth. CONCLUSIONS: Our system evaluated hydrogen water's potential against ischemia and found it potentially useful, which is consistent with expert assessment. Due to natural product's milder effects, large data sets are needed for evaluation, making manual measurement labor-intensive. Hence, our system offers a promising new approach for evaluating natural products.

Establishment and application of a new 4/6 infarct nephrectomy rat model for moderate chronic kidney disease.

PURPOSE: To develop a new 4/6 infarct nephrectomy (INx) model rat mimicking moderate chronic kidney disease (CKD) and to evaluate its application. METHODS: We modified the conventional 5/6 INx rat model to create the 4/6 INx model by ligating the renal artery branch to induce infarction of one-third of the left kidney after right kidney removal and compared biochemically and histologically both models. To demonstrate the application of the 4/6 INx model, the effects of a supplementary compound containing calcium carbonate, chitosan, palm shell activated charcoal etc., that is effective for both CKD and its complications, were compared between both models. RESULTS: Impairment of renal function in the 4/6 INx group was significantly more moderate than in the 5/6 INx group (P < 0.05). The 4/6 INx group showed less histological damage in kidney than in the 5/6 INx group. The supplementary compound did not improve CKD in the 5/6 INx group, but ameliorated elevation of blood urea nitrogen in the 4/6 INx group. CONCLUSIONS: We developed the 4/6 INx model, which is more moderate than the conventional 5/6 INx model. This model could potentially demonstrate the effectiveness of drugs and supplements intended to prevent CKD and its progression.

Firefly Rats: Illuminating the Scientific Community in Transplantation Research.

Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.

Establishment and application of a new 4/6 infarct nephrectomy rat model for moderate chronic kidney disease

Purpose: To develop a new 4/6 infarct nephrectomy (INx) model rat mimicking moderate chronic kidney disease (CKD) and to evaluate its application. Methods: We modified the conventional 5/6 INx rat model to create the 4/6 INx model by ligating the renal artery branch to induce infarction of one-third of the left kidney after right kidney removal and compared biochemically and histologically both models. To demonstrate the application of the 4/6 INx model, the effects of a supplementary compound containing calcium carbonate, chitosan, palm shell activated charcoal etc., that is effective for both CKD and its complications, were compared between both models. Results: Impairment of renal function in the 4/6 INx group was significantly more moderate than in the 5/6 INx group (P < 0.05). The 4/6 INx group showed less histological damage in kidney than in the 5/6 INx group. The supplementary compound did not improve CKD in the 5/6 INx group, but ameliorated elevation of blood urea nitrogen in the 4/6 INx group. Conclusions: We developed the 4/6 INx model, which is more moderate than the conventional 5/6 INx model. This model could potentially demonstrate the effectiveness of drugs and supplements intended to prevent CKD and its progression.

Impact of daily administration of blackberry extract on gerbil model of transient cerebral ischemia

Purpose: Blackberries are rich in polyphenols and are a human health food continuously consumed to improve health and reduce diseases caused by aging. Herein, we evaluated the effects of daily blackberry administration before and after transient cerebral ischemia in gerbils. Methods: Blackberry extract (BBE) was orally administered twice a day for two weeks to protect against ischemic events during continuous administration. On the seventh day after administration, the bilateral common carotid arteries were transiently occluded for 5 min. To verify its therapeutic effect, BBE was administered after ischemia using a similar protocol without pre-administration. In both experiments, the number of viable neurons in the CA1 region of the hippocampus was assessed seven days after ischemic treatment. Results: The number of neurons in the group treated with BBE before ischemia was higher than that in the group treated with distilled water (p = 0.0601), and similar to that in the control group. In the BBE administration experiments after ischemia, the number of neurons was significantly reduced compared to that in the control group (p < 0.0001). Conclusions: Continuous BBE intake is expected to prevent or ameliorate ischemic events such as transient cerebral ischemia.

A neural cell automated analysis system based on pathological specimens in a gerbil brain ischemia model

ABSTRACT Purpose: Amid rising health awareness, natural products which has milder effects than medical drugs are becoming popular. However, only few systems can quantitatively assess their impact on living organisms. Therefore, we developed a deep-learning system to automate the counting of cells in a gerbil model, aiming to assess a natural product's effectiveness against ischemia. Methods: The image acquired from paraffin blocks containing gerbil brains was analyzed by a deep-learning model (fine-tuned Detectron2). Results: The counting system achieved a 79%-positive predictive value and 85%-sensitivity when visual judgment by an expert was used as ground truth. Conclusions: Our system evaluated hydrogen water's potential against ischemia and found it potentially useful, which is consistent with expert assessment. Due to natural product's milder effects, large data sets are needed for evaluation, making manual measurement labor-intensive. Hence, our system offers a promising new approach for evaluating natural products.

Pre-Administration of Blackberry Extracts in Induced Ischemia Reperfusion Events in Rodents.

Blackberries are abundant in substances that have antioxidative and other effects, and technologies for enhancing the effectiveness of their incorporation into the body are being developed. The effectiveness of such substances has been investigated in various models, including rodent ischemia models. While a test substance can be administered either before or after an event, healthy foods are generally pre-administered prophylactically in experiments. Pre-administration may have the potential to elevate the blood concentration of the active substance sufficiently prior to the event and/or induce adaptive changes in the ischemic tolerance of the recipient through long-term pre-administration. Based on the recently reported 2-week pre-administration of blackberries in a rat model, we investigated the pre-administration of blackberry extracts in a hyperlipidemia model using Mongolian gerbils. We then discussed the effects of the pre-administration on the treated animals before an ischemic event.

A rat model of dual-flow liver machine perfusion system.

PURPOSE: As clinical liver perfusion systems use portal vein and artery flow, dual perfusion techniques are required even in small animal models in order to reproduce clinical setting. The aim of this study was to construct a new dual-flow perfusion system in rat model and optimized the oxygen supply to ensure the aerobic metabolization. METHODS: The dual-flow circuit was fabricated using rat liver and whole blood samples as perfusates. The oxygen supply was controlled according to the amount of dissolved oxygen in the perfusate. Perfusate parameters and adenosine triphosphate (ATP) levels were analyzed to evaluate organ function and metabolic energy state. Stored whole blood also tested the suitability as perfusate. RESULTS: Stored blood showed decrease oxygen delivery and liver function compared to fresh blood. Using fresh blood as perfusate with air only, the dissolved oxygen levels remained low and anaerobic metabolism increased. In contrast, with oxygen control at living body level, anaerobic metabolism was well suppressed, and tissue ATP content was increased. CONCLUSIONS: We developed a new dual-flow system that enable to reproduce the clinical settings. The perfusion system showed the possibility to improve the energy metabolic state of the perfused organ under appropriate partial pressure of oxygen.

A rat model of dual-flow liver machine perfusion system

Purpose: As clinical liver perfusion systems use portal vein and artery flow, dual perfusion techniques are required even in small animal models in order to reproduce clinical setting. The aim of this study was to construct a new dual-flow perfusion system in rat model and optimized the oxygen supply to ensure the aerobic metabolization. Methods: The dual-flow circuit was fabricated using rat liver and whole blood samples as perfusates. The oxygen supply was controlled according to the amount of dissolved oxygen in the perfusate. Perfusate parameters and adenosine triphosphate (ATP) levels were analyzed to evaluate organ function and metabolic energy state. Stored whole blood also tested the suitability as perfusate. Results: Stored blood showed decrease oxygen delivery and liver function compared to fresh blood. Using fresh blood as perfusate with air only, the dissolved oxygen levels remained low and anaerobic metabolism increased. In contrast, with oxygen control at living body level, anaerobic metabolism was well suppressed, and tissue ATP content was increased. Conclusions: We developed a new dual-flow system that enable to reproduce the clinical settings. The perfusion system showed the possibility to improve the energy metabolic state of the perfused organ under appropriate partial pressure of oxygen.

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-L-Lysine Induce Antibody Production as Vaccine Adjuvants.

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-ß. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.

Pharmacokinetics of hydrogen administered intraperitoneally as hydrogen-rich saline and its effect on ischemic neuronal cell death in the brain in gerbils.

Intraperitoneal administration of hydrogen (H2)-containing saline inhibited neuronal cell death in ischemic stroke in a number of animal models, but it is unknown whether H2 is absorbed from the abdominal cavity into the blood and reaches the brain. In this study, we investigated whether intraperitoneal administration of saline containing H2 inhibits neuronal cell death caused by cerebral ischemia and measured the concentration of H2 in the carotid artery and inferior vena cava (IVC). Gerbils were subjected to transient unilateral cerebral ischemia twice, and saline or H2-rich saline was administered intraperitoneally three or seven times every 12 hours. We evaluated the number of apoptotic cells in the hippocampus and cerebral cortex on day 3 and the number of viable neurons in the hippocampus and cerebral cortex on day 7. In addition, a single dose of saline or H2-rich saline was administered intraperitoneally, and blood H2 levels in the carotid artery and IVC were measured. On day 3 of ischemia/reperfusion, the number of neurons undergoing apoptosis in the cortex was significantly lower in the H2-rich saline group than in the saline group, and on day 7, the number of viable neurons in the hippocampus and cerebral cortex was significantly higher in the H2-rich saline group. Intraperitoneal administration of H2-rich saline resulted in large increases in H2 concentration in the IVC ranging from 0.00183 mg/L (0.114%) to 0.00725 mg/L (0.453%). In contrast, carotid H2 concentrations remained in the range of 0.00008 mg/L (0.0049%) to 0.00023 (0.0146%). On average, H2 concentrations in carotid artery were 0.04 times lower than in IVC. These results indicate that intraperitoneal administration of H2-rich saline significantly suppresses neuronal cell death after cerebral ischemia, even though H2 hardly reaches the brain.

H2 Inhibits the Formation of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) contribute to inflammatory pathogenesis in numerous conditions, including infectious and cardiovascular diseases, and have attracted attention as potential therapeutic targets. H2 acts as an antioxidant and has been clinically and experimentally proven to ameliorate inflammation. This study was performed to investigate whether H2 could inhibit NET formation and excessive neutrophil activation. Neutrophils isolated from the blood of healthy volunteers were stimulated with phorbol-12-myristate-13-acetate (PMA) or the calcium ionophore A23187 in H2-exposed or control media. Compared with control neutrophils, PMA- or A23187-stimulated human neutrophils exposed to H2 exhibited reduced neutrophil aggregation, citrullination of histones, membrane disruption by chromatin complexes, and release of NET components. CXCR4high neutrophils are highly prone to NETs, and H2 suppressed Ser-139 phosphorylation in H2AX, a marker of DNA damage, thereby suppressing the induction of CXCR4 expression. H2 suppressed both myeloperoxidase chlorination activity and production of reactive oxygen species to the same degree as N-acetylcysteine and ascorbic acid, while showing a more potent ability to inhibit NET formation than these antioxidants do in PMA-stimulated neutrophils. Although A23187 formed NETs in a reactive oxygen species-independent manner, H2 inhibited A23187-induced NET formation, probably via direct inhibition of peptidyl arginine deiminase 4-mediated histone citrullination. Inhalation of H2 inhibited the formation and release of NET components in the blood and bronchoalveolar lavage fluid in animal models of lipopolysaccharide-induced sepsis (mice and aged mini pigs). Thus, H2 therapy can be a novel therapeutic strategy for NETs associated with excessive neutrophil activation.

An organoid-based organ-repurposing approach to treat short bowel syndrome.

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.

A microsurgical technique for catheter insertion in the rat femoral artery.

PURPOSE: To modify a surgical catheterization method using the bent needle introducer in small animals. METHODS: Eight-week-old male Lewis rats were used in the study. A needle introducer was created by bending a 21G injection needle at 45°. The bent needle introducer was used for catheter insertion into the left femoral artery of the rats under anesthesia. As a control, a catheter was directly inserted into the blood vessel without the introducer. The insertion time of each method was measured. Blood pressure and heart rate were measured 24 h after catheter insertion using the telemetry system. RESULTS: Using the introducer, the catheter was successfully inserted within a short time in all rats. Without the introducer, a longer duration was required for catheter insertion. The frequency of the insertion with no catheter-based errors with the introducer tended to be higher than that without the introducer. The mean arterial pressure and heart rate 24 h after catheter insertion in each group were almost the same. CONCLUSIONS: We developed a surgical catheterization method using the introducer in small animals. This could potentially reduce the frequency of the insertion with catheter-based errors and insertion time.

Daily inhalation of hydrogen gas has a blood pressure-lowering effect in a rat model of hypertension.

A recent clinical study demonstrated that haemodialysis with a dialysate containing hydrogen (H2) improves blood pressure control in end-stage kidney disease. Herein, we examined whether H2 has a salutary effect on hypertension in animal models. We subjected 5/6 nephrectomised rats to inhalation of either H2 (1.3% H2 + 21% O2 + 77.7% N2) or control (21% O2 + 79% N2) gas mixture for 1 h per day. H2 significantly suppressed increases in blood pressure after 5/6 nephrectomy. The anti-hypertensive effect of H2 was also confirmed in rats in a stable hypertensive state 3 weeks after nephrectomy. To examine the detailed effects of H2 on hypertension, we used an implanted telemetry system to continuously monitor blood pressure. H2 exerted an anti-hypertensive effect not only during daytime rest, but also during night-time activities. Spectral analysis of blood pressure variability revealed that H2 improved autonomic imbalance, namely by suppressing the overly active sympathetic nervous system and augmenting parasympathetic nervous system activity; these effects co-occurred with the blood pressure-lowering effect. In conclusion, 1-h daily exposure to H2 exerts an anti-hypertensive effect in an animal model of hypertension.

Poor outcomes in carriers of the RNF213 variant (p.Arg4810Lys) with pulmonary arterial hypertension.

BACKGROUND: A variant of c.14429G>A (p.Arg4810Lys, rs112735431) in the ring finger protein 213 gene (RNF213; NM_001256071.2) has been recently identified as a risk allele for pulmonary arterial hypertension (PAH). PAH can be added as a new member of RNF213-associated vascular diseases, which include Moyamoya disease and peripheral pulmonary stenosis. Our aim was to identify the clinical features and outcomes of PAH patients with this variant. METHODS: Whole-exome sequencing was performed in 139 idiopathic (or possibly heritable) PAH patients. RESULTS: The RNF213 p.Arg4810Lys variant was identified in a heterozygous state in 11 patients (7.9%). Time-course changes in hemodynamics after combination therapy in the patients with the RNF213 p.Arg4810Lys variant were significantly poorer compared with those carrying the bone morphogenic protein receptor type 2 (BMPR2) mutation (n = 36) (comparison of changes in mean pulmonary arterial pressure, p = 0.007). The event-free rate of death or lung transplantation was significantly poorer in RNF213 p.Arg4810Lys variant carriers than in BMPR2 mutation carriers (5-year event-free rate since the introduction of prostaglandin I2 infusion, 0% vs 93%, respectively; p < 0.001). CONCLUSIONS: Idiopathic PAH patients with the RNF213 p.Arg4810Lys variant are associated with poor clinical outcomes even in recent times. Earlier consideration of lung transplantation might be required for RNF213 p.Arg4810Lys variant carriers who are developing PAH. Documentation of the RNF213 p.Arg4810Lys variant, as well as already known pathogenic genes, such as BMPR2, can provide clinically relevant information for therapeutic strategies, leading to a personalized approach for the treatment of PAH.