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Translational models

in vitro and in vivo models to study mechanisms, interventions and biomarkers
in metabolic diseases and their complications.

Metabolic Health Research (MHR) at TNO

translational model

MHR develops and performs in vitro and in vivo models to study mechanisms, interventions and biomarkers in metabolic diseases and their complications. These translational models include unique (humanized) transgenic mouse models, in vivo and in vitro models, read-out systems employing i.a. histology, biochemical assays, cell biology, molecular biology, immunology and inflammation markers. This preclinical research is strongly translational and aims to improve the predictability of efficacy and safety of pharmaceutical and food interventions by detailed knowledge of disease processes and mechanisms. MHR has a track record in applied science, study design, professional project management and quality systems. MHR offers customized services that can be tailored to customer needs by direct interaction of scientists of MHR and the customer.

NAFLD・NASH models,  Functional Biomarker

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Non-alcoholic steatohepatitis (NASH) is one of the most prevalent chronic liver diseases, which is closely associated with obesity, insulin resistance and dyslipidemia. These important features therefore need to be reflected in a preclinical model as well. Based on 25 years of research on translational metabolic disease models, TNO has developed the LDLR-/-.Leiden trangenic mice that accurately mimics the etiology and pathology of NASH and fibrosis in humans. By using a high-fat diet, with a macronutrient composition comparable to human diets (e.g., without added cholesterol), LDLR-/-.Leiden trangenic mice develop obesity, insulin resistance, adipose tissue inflammation, increased gut permeability with altered microbiota composition, and NASH with bridging fibrosis.

Diet-induced NAFLD/NASH/Liver Fibrosis models.
 ・Model-1: LDLR-/-.Leiden trangenic mice
 ・Model-2: ApoE3Leiden.hCETP tg mice
  Both models are the Type-II Diabetes like NASH / Liver Fibrosis model without chemical induction.

 Track records:
 ・Anti-inflammatory: Caspase-1 inhibitor Cenicriviroc; CCR2 & CCR5, Sodium salsalate, Icosabutate
 ・Anti-oxidative: Polyphenols, Anthocyanins, PUFAs, phytosterols
 ・PPAR activators: Rosiglitazone,GFT-505;Elafibranor,   Pioglitazone
 ・FXR activation: Obeticholic acid Volixibat
 ・Lipid-modulating: Ezetimibe, Rosuvastatin, EPA, DHA, Casein hydrolysate
 ・Mitochondria: miRNA 34a silencing, L-carnitine & Nicotinamide riboside,
 ・Translation to Human:Head to head with 1099 patients

□ Download; latest publication list
□ Recent poster at AASLD 2022:  The bispecific anti FGFR1/KLB agonist antibody bFKB1 attenuates non alcoholic steatohepatitis and atherosclerosis in Ldlr -/-.Leiden mice.

Diabetic Kidney Disease model

サンプルイメージ  Features for Diabetic Kidney Disease mouse model
 ・Progressive decline in renal function in setting of hyperglycemia,
    typically preceded by a period of glomerular hyperfiltration.
 ・Quick onset of Albuminuria
 ・Pathological changes in kidneys:
    > Glomerular basement membrane thickening
    > Mesangial matrix expansion and sclerosis
    > Tubulo-interstitial fibrosis
    > Arteriolar hyalinosis
 ・Diet induced (Human-like diet composition)
 ・The model shows hyperlipidemia, hyperglycemia and hypertension, albuminuria, decline in GFR and typical histological features of DKD
 ・Reference control: Combination of ACE inhibitor + SGLT2 ihibitor

Readout parameters
 ・Metabolic parameter: BW, F&W-intake, Glucose, Insulin, CHO, TG
 ・Function: Diuresis, Albuminuria, UACR, GFR by TD inulin clearance
 ・Pathology: Quantitative scoring of Glomerular and Tubular damage, Liver and Cardiac damage
 ・Optional: Proteomics/Metabolomics,Next generation sequencing, EM microscopy, PEMP analysis (NIPOKA)

Download Poster at ASN Kidney Week 2022
  A novel translational model of hypertension-accelerated diet-induced diabetic kidney disease with declining GFR and advanced pathology.

Sarcopenia, Frailty models

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The prevalence of sarcopenia is increasing and effective interventions are required to prevent or reverse age-related muscle loss. However, it often is challenging, expensive and time-consuming to develop and test the effectiveness of such interventions and translational animal models that are adequately mimicking the underlying physiological pathways are scarce. Strong predictors for the incidence of sarcopenia include a sedentary life-style and malnutrition. TNO has therefore recently developed a new and short (2 weeks) mouse model for muscle atrophy that combines caloric restriction with partial immobilization (of one hindleg). This combination model exhibits loss of muscle mass and function. Transcriptome analysis demonstrated that the underlying pathways of this combination model revealed more similarity with the human underlying pathways than aged mice. In addition to the combination models, we previously demonstrated a beneficial treatment effect in the CR alone model (van den Hoek AM et al., Metabolism 2019). In addition to the abovementioned models, TNO has several technologies (including AMS; Low dose 14C-alanine incorporation to enable high sensitive detection and tracing of protein build-up in muscle) available for muscle related readouts. In addition, we perform biomarkers research and can support companies with sensors/eHealth solutions in this therapeutic indication.

Model options; C57BL6 mice
 1: Caloric restriction model.
 2: Combination model: combining caloric restriction and immobilization.
     Regimen; either prophylactic, or therapeutic.

Readout parameters
 ・Echo-MRI: Lean body mass/body composition
 ・Functional tests: Grip strength, inverted screen, voluntary movement/physical activity
 ・Histology: Cell diameter; slow/fast fiber type, collagen
 ・Transcriptomics: Pathway analysis, super regulator prediction
 ・Protein signalling pathways: Pathway analysis (insulin, mTOR, AMPK, FOXO)
 ・Biochemical: Intramuscular triglyceride content
 ・AMS: Low dose 14C-alanine incorporation to enable high sensitive detection and tracing of protein build-up in muscle

Poster Download
□ Immobilization Combined with Caloric Restriction as Translational mouse model for Sarcopenia Expressing Key Pathways of Human Pathology.

Collaboration program
In addition, TNO is leader of a research consortium that focuses on a better understanding of sarcopenia, including the underlying mechanism. A unique human cohort is studied to further validate the translatability of our models and to identify biomarkers. In addition, male/female differences will be studied. Also we use advanced histological analysis in this project.

Future directions
We are currently looking to initiate a new research program in muscle, in which we would like to investigate the link between muscle health and cognition.

Fibrosis models

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 ・Lung Bleomycin-induced lung fibrosis model in mice
  (Feature:o.p.administration, low variation and motality)
 ・Skin Bleomycin-induced skin fibrosis model in mice.
 ・Liver CCL4-induced, Diet-induced model in mice
 ・Kidney UUO model in mice.
 ・in vitro fibrosis assay with the fibrosis patient samples
   Myoblast differenciation, Fibroblast proliferation, Migration

 Recent publication: Targeting the Wnt signaling pathway through R-spondin 3 identifies an anti-fibrosis treatment strategy for multiple organs

We're open to discuss;
 ・Collagen type analysis:Collagen 1α1, 3α1, 4α1, 5α1, 6α2.
 ・Signatutre analysis involving newly synthetised collagen

Reference
□ Collagen quantification in cell cultures and tissues.

Cardio Vascular and Metabolic disease models

サンプルイメージ APOE*3 LEIDEN MOUSE
This transgenic mice were generated by the introduction of the human apoe*3-Leiden and apoc1 genes. The primary effect of the dominant E*3-Leiden mutation is an impaired clearance of triglyceride-rich lipoproteins (chylomicron- and VLDL-remnants) caused by reduced affinity for the LDLR, whereas overexpression of APOC1 inhibits lipolysis. While normal wild-type mice have a very rapid clearance of apoB containing lipoproteins, E3L mice show an impaired clearance and are thereby mimicking the slow clearance in humans. As a consequence, E3L mice exhibit a human-like lipoprotein profile comparable to that of patients with familial dysbetalipoproteinemia (most of the circulating cholesterol is contained to (V)LDL particles), and develop atherosclerosis upon feeding with saturated fat and cholesterol. However, E3L mice (like wild-type mice) do not possess a Cholesteryl Ester Transfer Protein (cetp) gene, an essential component of human lipoprotein metabolism, and therefore these mice do not respond to HDL-modulating interventions.

APOE*3 LEIDEN.CETP MOUSE
This transgenic mice were generated by cross-breeding the E3L mice with CETP transgenic mice, which express the human cetp gene under control of its natural flanking regions. CETP transfers cholesteryl ester from HDL to the apoBcontaining lipoproteins in exchange for triglycerides, resulting in a more humanlike lipoprotein metabolism. As compared to the E3L mice, E3L. CETP mice are more prone to develop hyperlipidemia and atherosclerosis upon feeding a western type diet containing cholesterol, and very suited for (nutritional) interventions under human-like diet conditions.

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□ Models for Cardiovasucular and Metabolic diseases
□ Cardiovasucular Safety
□ Publication list


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