Age-related Changes In CD44 Levels And Its Role In Regulating Vascular Endothelial Cell Aging

Leukocyte differentiation antigen CD44 is a class of cell surface transmembrane glycoprotein type I,which is involved in intercellular,cell-extracellular matrix interaction,cell adhesion,cell migration and cell signal transduction.Cell senescence refers to the irreversible cell cycle arrest after a long period of population multiplication.Vascular endothelial cells,as the first line of defense of blood vessels,are one of the first cell types to age with aging.Body senescence is the result of cell senescence.It is considered that removing senescent vascular endothelial cells is an important strategy to maintain body health.However,a study published in Cell Metabolism reported that continuous or acute removal of senescent vascular endothelial cells is harmfulto health,which leads to the damage of blood tissue barrier and causes fibrosis in the liver and perivascular tissues,and subsequently exacerbates body damage.Therefore,rather than removing senescent vascular endothelial cells,the development of strategies to delay vascular endothelial cell senescence is a better choice for maintaining body health.In order to develop new interventions to delay vascular aging and prevent vascular lesions,it is important to deeply study the biological changes and molecular mechanisms of vascular endothelial cell aging.Autophagy is an evolutionarily conserved process of turnover of intracellular materials in eukaryotes.The aging damaged proteins or organelles are encapsulated by bilayer membrane autophagic vesicles and sent to lysosomes for degradation and recycling.A review published in the Cell in 2023 identified autophagy dysfunctionas a marker of aging.As the core mechanism of aging,autophagy dysfunction has become a hotspot and potential breakthrough point in anti-aging research.Endothelial cells are continuously exposed to circulating blood and are susceptible to various mediators,especially accumulation of toxic proteins and damaged organelles.Therefore,it is not difficult to understand that autophagy is particularly important for maintaining the normal function of vascular endothelial cells.Existing studies have shown that autophagy dysfunction is the core factor leading to vascular endothelial cell senescence and cardiovascular and cerebrovascular diseases.Our previous study found that CD44 is related to autophagy of vascular endothelial cells.However,whether CD44 mediates vascular endothelial cell senescence remains unclear.In this study,we found that the level of CD44 increased in an age-related manner in the human umbilical vein endothelial cells（HUVECs）and mouse aortic endothelial cells,suggesting a possible association between CD44 and senescence of vascular endothelial cells.Lentivirus-mediated silencing of CD44 showed decreased levels of aging markers P16 and P21 in HUVECs.Conversely,the levels of P16 and P21 proteins in HUVECs were significantly increased by lentivirus-mediated overexpression of CD44.CD44 knockout（CD44 KO）mice were constructed using CRISPR/CAS9 technology,and it was found that the activity of age-relatedβgalactosidase and the protein levels of aging markers P16 and P21 in aortic endothelial cells of CD44 KO mice were significantly reduced.It was confirmed that CD44 regulated vascular endothelial cell senescence.Further,CD44 silencing resulted in a significant increase in the number of LC3 puncta.the accumulation of LC3puncta may due to either excessive autophagy induction or impaired autophagosome degradation.To discriminate between these two possibilities,we used adenovirus-mediated expression of GFP-mRFP-LC3and found an increased number of both autophagosomes and autolysosomes in CD44-silenced HUVECs,similar to that observed in the EBSS or rapamycin-treated HUVECs,and was different from the Bafilomycin A1-treated cells.In vivo experiments showed that the levels of LC3 and P62 in the aorta of CD44 KO mice injected with chloroquinine（CQ）,an autophagy inhibitor,changed much more than those of Wild-type（WT）mice.These results indicated that CD44 negatively regulates autophagy in vascular endothelial cells.CD44 is divided into the extracellular domain（ECD）,transmembrane domain（TMD）and intracellular domain（ICD）.We overexpressed different truncated mutants of CD44 in HUVECs and determined that CD44ICD is required for its negative regulation.CD44 was knocked out in HUVECs using CRISPR/CAS9 technique and CD44ICD was added back.It was found that CD44ICD reversed the increase in autophagy activity caused by CD44 deletion.Next,we overexpressed CD44ICD in young HUVECs and found significant increases in age-relatedβ-galactosidase activity and levels of aging markers P16 and P21.CD44ICD endothelial specific overexpression(CD44ICD^flox/flox:Tek-Cre^+/-,hereafter referred to as CD44ICD^ECKI)mice were constructed using CRISPR/PRO technology.The lifespan of CD44ICDECKI mice was found to be significantly lower than that of litter control mice(CD44ICD^flox/flox:Tek-Cre^-/-,WT).Finally,we treated vascular endothelial cells with rapamycin and trehalose,and found that activation of autophagy effectively reversed cell senescence caused by CD44ICD overexpression.These findings suggest that CD44 inhibits autophagy through its ICDs,leading to"loss of youth"in vascular endothelial cells.In this study,we systematically studied the changing trend of CD44 level in vascular endothelial cells during aging,revealing the"pivotal role"of CD44 in mediating autophagy disorder and aging of vascular endothelial cells through its intracellular segment ICD,providing a new experimental basis for clarifying the molecular mechanism of vascular endothelial cell aging.At the same time,it provides new clues and targets for delaying vascular aging and preventing cardiovascular diseases.