The Effect And Mechanism Of Adenosine And Its Receptors On Cardiovascular Tissue Engineering

In recent years, the incidence and mortality of cardiovascular diseases has increased year by year, and each year a large number of patients require bypass surgery, leading to a high clinical demand for tissue-engineered blood vessels(TEBV) less than 6 mm in diameter. However, small-diameter TEBV are prone to thrombosis and intimal hyperplasia, resulting in failed transplant surgeries. Rapid endothelialization during the early stages is the key to maintaining the patency of grafted small-diameter TEBV. Early endothelialization can be achieved by coating the inner surface of vascular grafts with specific antibodies or molecules to specifically capture EPCs in the circulation, but there always remain limitations. CD34 antibody coating could promote rapid endothelialization of grafts, whereas intimal hyperplasia is increased at profoundly 1 month after implantation. The main reason is that the local microenvironment of TEBV could induce the pathological proliferation of smooth muscle cell(SMC), but not suitable for EPCs survival. In addition, the patients need vascular bypass grafting always accompanied with diabetes and other basic diseases, high glucose and high glucose induced oxidative stress in vascular tissue engineering makes the local micro environment worse, further damaging homing EPCs. Therefore, besides choosing appropriate molecules to promote stem cell mobilization and selectively capture EPCs, Optimizing the microenvironment is also an important research target to improve the patency of TEBV.Adenosine is an endogenous nucleoside that is distributed in cells throughout the human body. Under ischemic injury, the local adenosine concentration will increas e to protect cells and promote angiogenesis; therefore adenosine is called as “retaliatory metabolite”. The microenvironment of transplanted TEBVs is a Hypoxia, ischemia and inflammation increased environment. Adenosine could promote TEBV Long term patency through optimizing the microenvironment. Thus we used adenosine and its receptors as research tools, to study the effect and mechanism of optimizing the microenvironment on cardiovascular tissue engineering.1. Adenosine optimizes the microenvironment of TEBV through regulation of energy metabolismBone marrow hematopoietic stem cells are in the resting and undifferentiated state under physiological conditions to ensure the stability of the amount of blood cells in the bone marrow and peripheral blood circulation. Mondal et al. have found that the maintenance of the resting state is mediated by adenosine deaminase growth factor A(Adgf-A), which reduces the adenosine concentration to an extremely low level. Our study demonstrated for the first time that adenosine promoted EPC mobilization and homing via energy conversion, achieving rapid endothelialization of blood vessels at an early stage. In addition, adenosine also optimized the microenvironment of the tissue-engineered blood vessels to improve the patency of the adenosine-modified tissue-engineered blood vessels. However, adenosine is quickly taken up by cells. We prepared adenosine loaded CS/β-CD nanoparticles and constructed adenosine modified TEBV by using layer-by-layer assembly. The adenosine molecules on the inner surface coating of the TEBV could be released quickly to promote EPC mobilization and homing to achieve rapid endo thelialization. And the CS/β-CD nanoparticles loaded with adenosine slowly released the adenosine locally to optimize the local microenvironment of the vascular grafts for long periods of time and maintain the vascular patency.2. Adenosine receptors regulate crosstalk between monocyte / macrophage and EPCsTEBV scaffold materials mostly comprise allograft acellular material or polymers, such as poly(lactic-co-glycolic) acid(PLGA). The entry of these materials into the body can inevitably cause bodily inflammation. Inflammation is considered the most important mechanism of thrombosis and intimal hyperplasia. However, Breuer et al. found that tissue-engineered vascular grafts could transform into mature blood vessels via the inflammation-mediated process of vascular remodeling. Therefore, the appropriate regulation of inflammatory responses could be an effective method of maintain ing TEBV patency. Our results showed that endothelial progenitor cells(EPCs) have two facets that are well aligned with the Chinese yin-yang theory: a yin(proinflammatory) aspect and a yang(proangiogenic) aspect. Acellular materials will not directly cause acute and severe inflammatory responses but will activate proinflammatory molecule expression in homing EPCs, gradually promoting the polarization of the monocyte/macrophage to the M1 type and eventually leading to TEBV failure. Adenosine A2 a receptor activation promotes the secretion of more proangiogenic factors, inducing EPC migration and mobilization. Moreover, adenosine A2 a receptor activation inhibited monocyte/macrophage polarization to the M1 type through the down-regulation of EPC proinflammatory molecules, such as ICAM-1, VCAM-1 and E-selectin. In summary, EPC proinflammatory molecules can be used as effective indicators for the evaluation of the long-term biological effect of materials, and the EPC-monocyte/macrophage interaction could be used as a new target for the design of tissue engineering material. If the materials cannot meet this requirement, we can control the drug release to activate certain targets, such as the adenosine A2 a receptor.3. Endogenous adenosine contributes to the patency rate of TEBVThe porosity and pore size of tissue-engineered material are closely related to the survival, proliferation, and differentiation of the cells it contains. Open porous and interconnected networks are conducive to the exchange of nutrients an d oxygen between cells in the biomaterial and the external environment. It is reported that 30 to 40 μm is the smallest pore size of biomaterial for TEBVs that can maintain adequate nutrients and oxygen exchange. However, a greater porosity and pore size will reduce the structural stability of the biomaterial. Grafted TEBVs must withstand a high fluid shear stress and scouring force from the blood. Thus, an unstable structure of the biomaterial will readily cause clogging, deformation, and even rupture of T EBVs, resulting in serious consequences. A few scholars consider the appropriate pore size for the inner layer of TEBVs to be less than 24 μm, which is smaller than the minimum size required for maintaining material exchange. Therefore, the local microenvironment of TEBVs exhibits a significant lack of oxygen and nutrients before the ingrowth of capillary vessels from the outer layer, which is not conducive to cell survival. Phosphorus is an important component of cell membranes, ATP, and nucleic acids, and large amounts of phosphorus ions(PI) are released after cell death. Because a lower porosity and smaller pore size are insufficient for the timely exchange of metabolic wastes, the concentration of PI will significantly increase in the local microenvironment of grafted TEBVs. The increase in PI concentration is closely related to the development of cardiovascular diseases. Additionally, an increased concentration of PI can contribute to cellular energy metabolism and stimulate ATP production. These processes will further increase the metabolic burden of cells in the ischemic-hypoxic environment of the graft material, exacerbating cell injuries and death. We found that klotho could change the response pattern of infiltrated monocytes/macrophages to the high extracellular PI, reducing the energy metabolism level to maintain cell survival and inhibiting high PI-inducing apoptosis. A decline in energy metabolism of monocytes/macrophages resulted in the degradation of intracellular ATP and a massive release of adenosine. Increased adenosine could facilitate the mobilization of endothelial progenitor cells(EPCs) through improving EPC energy metabolism in bone marrow. Meanwhile, klotho promoted the homing of increasing EPCs, which contribute to the rapid endothelialization of TEBVs via differentiating into endothelial cells and paracrine secretion. Next we modified Klotho protein to the TEBV through layer-by-layer assembly. At 6 months post-grafting, the patency rate of klotho-modified TEBVs reached 90% and good endothelialization was also observed.4. The mechanism of adenosine protects EPCs in diabetes mellitusPatients who require vascular replacement therapy always have primary disease and adenosine modified TEBV has failed to achieve good performance in the va scular replacement therapy of diabetes mellitus. Until now, no successful construction of TEBV in the replacement of diabetic vessels has been reported. Autophagy is an important physiological phenomenon in multicellular organisms. During energy starvation or environmental stress, autophagy plays an important role in the maintenance of cellular homeostasis. It is well known that autophagy plays an important role in cellular homeostasis through the degradation and recycling of organelles such as mitochondria or the endoplasmic reticulum(ER) that are closely related to the pathogenesis of diabetes. Our study found that the MMP of EPCs in patients with diabetes mellitus was higher than that in the healthy subjects, but the level of autophagy was much lower. The imbalance between autophagy and energy metabolism might be an important reason for cell injury and death in diabetes mellitus. Interestingly, we found that high extracellular adenosine decreased the level of cell energy metabolism and increased EPC autophagy. Adenosine can maintain the survival and differentiation of EPCs grown on biomaterials by increasing high glucose-inhibited EPC autophagy and maintaining cellular energy metabolism. Thus adenosine can be used as an important target for the constructio n of TEBVs in diabetes mellitus.5. Construction of anti-high glucose injury TEBVsAdenosine has the functions of cell protection, EPC mobilization and homing, optimizing the microenvironment, but adenosine modified TEBV always failed in diabetes mellitus. Until now, no successful construction of TEBV in the replacement of diabetic vessels has been reported, a finding that can be attributed to two aspects. First, in diabetic patients, the functions of peripheral blood stem cells, such as migration, paracrin e functions, and differentiation that are related to the cells’ proangiogenic capability, are seriously damaged, leading to their failure in facilitating rapid endothelialization after homing to the inner surface of TEBVs. Second, the microenvironment of hyperglycemia in the diabetic patients can lead to exacerbation of the inflammatory response after TEBV transplantation and further damage to the stem cells that have homed to the graft. Ideal coating drugs of TEBVs used for diabetes must have at least four functions, including capturing stem cells, restoring the damaged functions of stem cells, protecting against high-glucose injury, and optimizing the microenvironment. Adenosine kinase(ADK) is a nucleoside kinase that catalyzes adenosine to form adenosine 5-triphosphate(ATP). Studies have shown that ADK upregulation accelerates cellular damage and death under ischemic conditions, and ADK inhibition helps to maintain cellular energy metabolism to fight against disease hazards. Furthermore, ADK deficient cells can sustainably release adenosine, which improves the local microenvironment through its functions such as those in inflammatory regulation, cell protection, and proangiogenesis. All above support the possibility of ADK as a functional target of stem cells under diabetic conditions. Thus we proposed an aptamer-si RNA chimeras modified TEBV that can maintain a satisfactory patency in diabetes. This TEBV consisted of two parts, CD133-adenosine kinase(ADK) chimeras and TEBV scaffold. CD133-ADK chimeras could selectively capture the CD133-positive cells in the blood, and the captured cells internalized the bound chimeras to achieve RNA self-transfection. Subsequently, CD133-ADK chimeras were cut into ADK si RNA by Dicer, resulting in depletion of ADK. ADK deficient cell may act as a bioreactor that sustainably releases adenosine. To reduce non-specific RNA transfection, we increased the proportion of HAu Cl4 during the material preparation, through which the transfection capacity of PEI/PEG-Au NPs was significantly decreased, and the ability of TEBV to resist tensile and liquid shear stress was greatly enhanced. PEG and 2’-OMe modification was also used to enhance the in vivo stability of RNA chimeras. Our study provides a new strategy for RNA application in tissue engineering materials.