Project Summary/ Abstract The growing prevalence of multifocal cerebral microhemorrhages (CMHs), also known as "cerebral microbleeds," is a significant and newly recognized problem in the aging population. These CMHs are major contributors to vascular cognitive impairment (VCI) and gait and balance disorders, which are among the most common causes of falls in older adults. Hypertension is the primary risk factor for CMHs in older adults, which are associated with the rupture of small intracerebral vessels and progressive impairment of neuronal function. Despite affecting over 50% of older individuals, the pathogenesis of CMHs remains obscure, and no therapeutic interventions are currently available for their prevention. The objective of the present proposal is to elucidate the specific age-related mechanisms that lead to increased cerebromicrovascular fragility and identify potential targets for prevention of CMHs using innovative preclinical models. Results from recent studies provided insights into the cellular mechanisms by which aging may increase cerebromicrovascular fragility and promote development of CMHs: increased presence of senescent endothelial cells in the cerebral microcirculation. Our central hypothesis is that the accumulation of senescent endothelial cells in the cerebral microcirculation, caused by aging, results in a microvascular phenotype that is prone to fragility. Senescent cells release activated matrix metalloproteinases and other paracrine factors ("senescence- associated secretory phenotype" or SASP), which promote structural maladaptation to high pressure, degradation of collagen and remodeling of extracellular matrix, thus creating loci of least resistance that can easily burst upon induction of hypertension. We will combine advanced imaging techniques and cutting-edge transcriptomics to investigate microvascular structure and gene expression in senescent cells in the cerebral microcirculation of aged mice. Our prediction, based on our working hypothesis, is that elimination of senescent cells, either through genetic manipulation (p16-3MR mouse model) or by pharmacological means will protect the structural integrity of cerebral vessels preventing the development of CMHs. Specific Aims: 1) Determine the causal link between increased presence of senescent cells in the microcirculation and genesis of CMHs in aged mice. 2) Determine whether induction of accelerated senescence exacerbates CMHs in young mice, mimicking the aging phenotype. 3) Determine how senescence promotes a pro-fragility phenotype in cerebral microvessels. Together, the proposed studies will identify a fundamental mechanism responsible for age-related exacerbation of CMHs, and the resulting vascular-induced neurological deficits. The increased presence of senescent cells in the microcirculation and consequential degradation of cerebrovascular structural integrity are key contributors to this phenomenon. By uncovering these mechanisms, our research will...