Aging Reprograms The Hematopoietic-Vascular Niche To Impede Regeneration And Promote Fibrosis

After acute injury,mammalian organs can undergo facultative regeneration to replace damaged tissues.The regenerative ability plays a vital role in maintaining the homeostasis of multicellular organisms.However,regenerative capacity in most mammals is limited after chronic injury.With the development of industrialization,more and more factors in our life can stimulate the occurrence of chronic tissue damage,and the final pathological results of these chronic tissues are usually manifested as fibrosis.Fibrosis is generally defined as the excessive deposition of extracellular matrix（ECM）components,which leads to scar tissue formation,and ultimately organ dysfunction and,in severe cases,organ failure and death.Progressive structural remodeling（fibrosis progression）can be seen in almost all fibrotic tissues and organs.According to statistics,up to 45%of all deaths are related to fibrosis.Organ fibrosis has been considered the main cause of incidence rate and mortality worldwide.In addition,in recent years,organ fibrosis has become the key to the development and treatment of malignant tumors due to the important influence of organ fibrosis on the occurrence,invasion and metastasis of tumors and drugs delivery to tumors.However,there is still no effective treatment for fibrosis.Especially for organs with end-stage fibrosis,the only treatment is organ transplantation.However,due to the limitation of limited donors and high price,this method can not be an effective method to solve the problem of organ fibrosis.Therefore,the research on the general mechanism of organ fibrosis and the development of efficient and economic treatment methods have become the urgent scientific problems in the field of biomedical research.Traditional regenerative research is mostly limited to the repair of organ parenchymal cells.Our study expands this concept by suggesting that the"circulating niche"composed of blood vessel cells and blood cells is similar to the"soil"in the organ repair process and can regulate the repair of parenchymal cells as"seeds".The circulating niche has a strong duality.The niche with different activation states can lead to two completely different results in the damaged organs,namely,functional regeneration or fibrosis（scarring）.Therefore,by analyzing the circulating niche,we try to find the"switch",that is,the key node molecules in the process of fibrosis.Through targeted editing,we can inhibit fibrosis and turn on the organ repair and regeneration process.The blood vessels in an adult body are 90,000km long,and their lumens are covered with 10 trillion to 6 trillion vascular endothelial cells,which permeate every cell in every organ.Due to its special location,vascular endothelial cells act as a"bridge"for material exchange and information communication between blood cells and parenchymal cells.Besides barrier function,they also regulate the physiological functions of peripheral cells through their strong paracrine and autocrine functions.Based on the above assumptions,we used pneumonectomy（PNX）to compare the lung alveolar regeneration and fibrosis in wild-type mice at different ages,including restoration of pulmonary function,epithelial architecture,and amounts of hydroxyproline.Compared with mice at the age of 2,3,and 6 months,the alveoli of20-month-old mice collapsed and the lung structure was disordered,and the pulmonary function was greatly inhibited.Old mice showed marked suppression of alveolar regeneration and increased fibrosis after PNX.In view of this phenomenon,we isolated pulmonary capillary endothelial cells（PCECs）from 2-month-（young）and 20-month-old（old）mice after PNX for transcriptome sequencing.On the basis of the transcription profiles,we compared the expression of EPCR that is selectively expressed in ECs.NRP1 and HIF2αare induced in ECs by pathological conditions.EPCR expression was lower in old PCECs after PNX than in 2-,3-,and 6-month-old mice.By contrast,expression of NRP1 and HIF2a was elevated in the PCECs of old mice after PNX.NRP1 is co-receptor for several cytokines.To dissect its functional contribution in aging-related fibrosis,we generated mice deficient of Nrp1 specifically in EC(Nrp1^{i DEC/i DEC}).Collagen,as the main component of extracellular matrix,can destroy the normal structure of tissue and inhibit the regeneration ability of injured organs.After PNX,old Nrp1^{i DEC/i DEC} mice exhibited less collagen deposition,enhanced alveolar regeneration,reduced senescence and fibroblast activation,increased propagation of type 2 alveolar epithelial progenitor cells,and lower number of perivascular neutrophils,compared with those of old control mice.The results show that deletion of Nrp1 in endothelial cells can inhibit fibrosis.EPCR neutralizing antibody 1560 abrogated the beneficial effects observed in old Nrp1^{i DEC/i DEC} mice.The transcriptome results also showed that Nrp1 knockout inhibited the expression of HIF2αand promote the expression of EPCR.Therefore,we found that aging can make endothelial cells reprogrammed,and elevated Nrp1 and HIF2αcan inhibit the expression of EPCR,thus promoting aging organ fibrosis and inhibiting its regeneration ability.As an important cell type in immune response,macrophages phagocytose and clear apoptotic cells as a part of their normal homeostatic function in tissues,when they encounter invading organisms or necrotic debris following injury,they become activated by endogenous dangers signals.These activated macrophages produce anti-microbial mediators like reactive oxygen and nitrogen species and proteinases that help kill invading pathogens,which helps restore tissue homeostasis.They also produce a variety of inflammatory cytokines and chemokines,matrix metalloproteinases and other inflammatory factors that help drive inflammatory and anti-microbial responses forward.The role of macrophages in wound healing response has two sides and always is regulated by the niche.There was little deposition of platelets in the young wild-type mouse lungs after PNX.By contrast,platelets associated with macrophages in the old wild-type mouse lung,forming perivascular cellular rosettes.Then we studied whether macrophages were regulated by reprogrammed endothelial cells during fibrosis in aged mice.We generated mice deficient of HIF2αspecifically in EC(HIF2α^{i DEC/i DEC}).Compared with the lungs of old control mice,recruitment of CXCR4⁺macrophages was decreased in old HIF2α^{i DEC/i DEC} mouse lungs after PNX,which was reversed by antibody 1560.Proliferation of alveolar epithelial progenitor was enhanced in old HIF2α^{i DEC/i DEC} mice after PNX,and 1560 blocked the increase of cell proliferation.Pulmonary macrophages were isolated from old control and HIF2α^{i DEC/i DEC} mice after PNX.The results showed that TIMP1 differed markedly between old HIF2α^{i DEC/i DEC}and control mice.The fibrogenic function of TIMP1 was then defined.The data suggested TIMP1 interacts with integrinβ1 to stimulates fibroblast activation.To unravel the role of CXCR4⁺TIMP1^high macrophages in aging induced pulmonary fibrosis,we utilized an adoptive macrophage transfer approach.Transplantation of Timp1-null monocytes promoted the recovery of pulmonary function and decreased collagen deposition in pneumonectomized old mice.As such,reprogramming of NRP1-HIF2a-EPCR pathway in the EC of old organs recruits pro-fibrotic CXCR4⁺TIMP1^high macrophages,impeding regeneration and stimulating fibrosis.Platelets are not only involved in hemostasis and thrombosis formation,but also play an important role in inflammatory response.Following vascular insult or injury,platelets become activated in the blood resulting in adhesion to the exposed extracellular matrix underlying the endothelium,formation of a platelet plug,and finally formation and consolidation of a thrombus consisting of both a core and shell.By secreting some pro-inflammatory factors,such as interleukin,platelets interact with macrophages to participate in some physiological processes.There was little deposition of platelets in the young wild-type mouse lungs after PNX.By contrast,platelets associated with macrophages in the old wild-type mouse lung,forming perivascular cellular rosettes.We hypothesized that reprogramming of NRP1-HIF2a-EPCR pathway in the EC of old organs induces crosstalk between platelets and macrophages,impeding regeneration and stimulating fibrosis.To assess the contribution of platelet IL-1a,we generated mice with platelet-specific deletion of IL-1a(IL1α^ΔPlt/ΔPlt).Given that EPCR-neutralizing antibody 1560induces fibrotic-like phenotype in young mice after PNX,we used 1560 to treat 3-mon IL1α^ΔPlt/ΔPlt mouse to establish the correlation between EPCR and platelet IL-1a.Deleting Il1a in platelet blocked the formation of platelet-macrophage rosette and reduced the pulmonary expression of pro-fibrotic TIMP1 in mice treated with EPCR m Ab.So it is likely that suppression of EPCR in old ECs leads to platelet IL-1a production and activation of pro-fibrotic TIMP1+macrophages,causing fibrotic phenotypes.Then we used a platelet adoptive transfer model to test the contribution of reprogramed platelet(IL1α^ΔPlt/ΔPlt)in protecting against pulmonary fibrosis induced by aging.Thrombopoietin（Thpo）is a major regulator of platelet numbers.Platelet number is decreased in Thpo^-/-mice by 95%as compared to wild-type mice.Compared with old mice transferred with Il1a^+/+platelets,old mice injected with IL1α^-/-platelets exhibited reduced TIMP1 expression and less collagen deposition in tested organs.These results indicate that targeted editing platelets can promote the transformation from fibrosis to regeneration after lung injury in aging mice.The above results found in lung were also verified in liver and kidney.Aged organs are susceptible to fibrosis after injury.Here,we show that aging-reprogramed crosstalk between platelets,macrophages,and vascular ECs results in loss of regenerative capacity and fibrogenesis.In aged lung,liver,and kidney,induction of Nrp1-HIF2αsuppresses anti-thrombotic and anti-inflammatory EPCR pathway,leading to formation of pro-fibrotic platelet-macrophage rosette.Activated platelets via supplying interleukin 1a synergize with endothelial-produced angiocrine chemokine to recruit fibrogenic TIMP1^high macrophages.In mouse models,genetic targeting of endothelial Neuropilin-1-HIF2a,platelet interleukin 1a,or macrophage TIMP1normalized the pro-fibrotic hematopoietic-vascular niche and restored the regenerative capacity of old organs.Targeting of aberrant endothelial node molecules might help propel "regeneration without scarring" in the repair of multiple organs.