Aging, Healthy Ageing, Inflammation
Conditions
Keywords
mitochondrial dysfunction, skin-derived fibroblasts, mtRNA, PBMCs, senescence, inflammaging
Brief summary
The MIRACLE study aims to investigate age-related mitochondrial dysfunction, mitochondrial RNA (mtRNA) release, inflammation, and cellular senescence in adult participants across three age groups. Skin-derived fibroblasts and peripheral blood mononuclear cells (PBMCs) will be isolated from skin biopsy and blood samples to characterize age-related cellular and molecular changes and to test experimental therapeutic strategies identified in preclinical studies. Serum, plasma, and whole-blood RNA will be used for protocol-defined analyses of circulating inflammatory mediators and systemic transcriptional signatures related to inflammation, type I interferon activation, mitochondrial stress response, immune aging, and senescence-associated pathways.
Detailed description
Mitochondria are crucial for ATP production and intracellular signaling in higher eukaryotic cells. These organelles contain genetic material that reflects their bacterial ancestry, including mitochondrial RNA (mtRNA). Under normal conditions, mtRNA is tightly confined and processed within mitochondria, ensuring the proper synthesis of proteins required for oxidative phosphorylation. Recent evidence suggests that mitochondrial stress may promote the leakage of mitochondrial components, including mtRNA. Once released into the cytosol, mtRNA can be sensed by cytosolic pattern recognition receptors (PRRs), thereby activating signaling pathways that promote the production of pro-inflammatory cytokines. Although these mechanisms have been investigated mainly under conditions of acute stress, the specific contribution of mitochondrial dysfunction and mtRNA release to chronic low-grade inflammation and cellular senescence during physiological aging remains incompletely understood. This represents an important knowledge gap in the understanding of molecular processes that may contribute to age-related inflammation and functional decline. The MIRACLE project includes a broade series of preclinical in vitro and in vivo experiments aimed at clarifying the mechanisms linking mitochondrial dysfunction, mtRNA release, inflammatory pathway activation, and cellular senescence during aging. These experimental activities are designed to define the biological pathways involved and to identify potential strategies capable of modulating mtRNA-associated inflammatory and senescence responses. Within this broader framework, the present human study is intended to corroborate and extend the preclinical findings in humans. To this aim, adult participants across different age groups will be enrolled, and skin biopsy and blood samples will be collected to obtain skin-derived fibroblasts, peripheral blood mononuclear cells (PBMCs), serum, plasma, and whole-blood RNA. Skin-derived fibroblasts will provide an accessible primary cell model for the investigation of age-related cellular and molecular changes. Fibroblasts isolated from participants of different ages will be used to assess mitochondrial function, mtRNA release, inflammatory signaling, and markers of cellular senescence. PBMCs collected from the same participants will be analyzed as a complementary blood-derived cellular model to evaluate systemic immune and inflammatory features, including immunosenescence-related signatures. Serum and plasma samples will be used to measure circulating inflammatory mediators and senescence-associated factors, while whole-blood RNA will be used to assess systemic transcriptional signatures related to inflammation, type I interferon activation, mitochondrial stress response, immune aging, and senescence-associated pathways.
Interventions
PROCEDURESkin biopsy and venous blood sampling
A single 3-4 mm punch biopsy of forearm skin is performed under local anesthesia (lidocaine 1% with epinephrine 1:100,000) for primary dermal fibroblast isolation. Venous blood is collected by standard phlebotomy from the antecubital fossa (43 mL total volume per participant): 5 mL in SST tube for serum separation, 3 mL in EDTA tube for plasma separation, 32 mL in EDTA tubes for PBMC isolation by density gradient centrifugation, and 3 mL in Tempus Blood RNA tube for whole-blood RNA stabilization. All procedures are performed at a single visit at enrollment. Samples are processed for biospecimen-based ex vivo molecular and cellular analyses; no investigational product is administered to participants.
Sponsors
Mario Negri Institute for Pharmacological Research
Study design
Allocation
NON_RANDOMIZED
Intervention model
PARALLEL
Primary purpose
BASIC_SCIENCE
Masking
NONE
Intervention model description
Cohort, cross-sectional
Eligibility
Sex/Gender
ALL
Age
18 Years to 90 Years
Healthy volunteers
Yes
Inclusion criteria
* Male and female * Age between 18 and 90 years (stratified in three groups: young, middle-aged, elderly) * Written informed consent
Exclusion criteria
* Inability to understand the potential risk and benefits of the study * Legal incapacity * Subjects who have taken antibiotics, anti-inflammatory drugs, or antihistamines within the past 7 days * Diagnosis of diabetes mellitus * Use of anticoagulant medications * Any subject with a contraindication to the mini-invasive biopsy procedure
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Age-dependent differences in cytosolic release of mitochondrial RNA (mtRNA) in primary human fibroblasts | Day 1 (at enrollment) | Assessment of release of mtRNA in the cytosol of primary fibroblasts isolated from study participants across age groups. Cytosolic mtRNA levels assessed through analysis of the mean fluorescence intensity (MFI) per cell by immunofluorescence. |
| Age-dependent differences in mitochondrial function in primary human fibroblasts | Day 1 (at enrollment) | Characterization of mitochondrial function parameters in primary fibroblasts isolated from study participants across age groups. Mitochondrial function assessed through analysis of mitochondrial Complex I activity in nmol/min/mg protein by functional ELISA. |
| Activation of inflammatory pathways in primary human fibroblasts | Day 1 (at enrollment) | Assessment of inflammatory pathway activation markers in fibroblasts isolated from participants across age groups. Inflammatory pathway activation assessed through Interleukin-6 concentration in skin-derived fibroblast culture supernatant in pg/mL |
| Activation of senescence pathways in primary human fibroblasts | Day 1 (at enrollment) | Assessment of cellular senescence markers and senescence-associated pathway activation in fibroblasts isolated from participants across age groups. Senescence pathway activation assessed as percentage (%) of Senescence-associated β-galactosidase (SA-β-gal)-positive cells per microscopic field. |
| Comparison of fibroblast findings with PBMC-derived parameters from the same participants | Day 1 (at enrollment) | Integration and comparison of fibroblast-derived molecular data with corresponding PBMC-derived measurements obtained from the same participants as described above. |
Countries
Italy
Contacts
CONTACTLuca Perico, PhD
CONTACTMiriam Rigoldi, MD
STUDY_DIRECTORGiuseppe Remuzzi
Istituto Di Ricerche Farmacologiche Mario Negri
Outcome results
None listed