| BackgroundSickle cell disease (SCD) is the common blood disorder with autosomal dominant inheritance, which is characterized by chronic insistent inflammation. This hemoglobinopathy manifests as a debilitating disease characterized by hemolytic anemia, chronic oxidative stress, severe infections, stroke, and pulmonary complications beginning in early childhood and lasting throughout life. Sickle red cells are inflexible with less deformability. They are easier to get broken and in circulation or endocytosed by macrophages, and hemoglobin inside can be released into blood resulting in hemolysis. Masses of cell-free hemoglobin in circulation will lead to the release of freeoxygen radicals and relevant oxidative response. The viscosity of the blood is obviously increased with slower flow. The factors stated above can induce vascular congestion and tissue inflammatory damage.High intensity lipoprotein (HDL) was used to be considsered as anti-inflammatory properties and being mainly responsible for reverse cholesterol transport (RCT) to inhibit arterosclerosis (AS) and prevent cardiovascular disease. The role of HDL to inflammation depends on the properties of its associated particles. The protein conponents in HDL are variable to follow the worse inflammatory environment. Reduced ApoA-land PON1are capable to resist inflammation and oxidative stress, while Interleukin (IL), Serum Amyloid A and other proinflammatory factors are increased. These make HDL convert from anti-inflammation to become proinflammatory (p-HDL). It impairs the RCT function and limited to prevent LDL oxidation. P-HDL is a part of proinflammatoy factors to induce the development of cardiovascular disease and angiopathy. P-HDL is an important marker focus by cardiovascular clinic and basic study. It is therefore insufficient for merely quantified HDL to meet the demand on clinic and research job. Currently, many experts agree on the opinion to quantify and qualify HDL. It is profound to quantify p-HDL to assess cardiovascular risk. To determine the p-HDL level in plasma, it is used to take precipitation of Apo B containing lipoproteins to get the HDL supernatant and HDL was oxidized by Dichlorofluorescein (DCFH) and CuCl2(or oxLDL). The data of p-HDL was got by plate-reading as previously. It is admitted that precipitation of apoB is easy and brief, but the supernatant still hold some proinflammatory proteins that have a influence on p-HDL data. Plasma cell-free hemoglobin and xanthine oxidase (XO) can be increased in SCD. As mentioned above, lots of hemoglobin was released into blood from broken sickle red cells and caused oxidative tissue injuries. XO is considered as strong and non-specific oxidant. It holds the ability to catalyze many substance to produce superoxide anions and hydroperoxides to aggravate oxidative stress as electronic donor. Albumin also impacts its influence on oxidation, although the controversy is on its orientation (prevention or promotion) in previous study. But it is definite that albumin is able to interfere with oxidation anyhow. Thus to determine the influence of the supernatant on p-HDL measurement, we purify HDL in plasma of SCD patients by Anion Exchange-High Performance Liquid Chromatograph (AE-HPLC) and detect p-HDL level to compare with ApoB precipitation assay. However, it is still not well understood on the mechanism that HDL converts into p-HDL. As described previously, remarkable elevation of cf Hb in plasma is the main pathophysiological process in SCD. And heme is increased greatly as well as cf Hb for hemolysis. heme itself is a strong oxidant and strengthen the oxidative injuries of cf Hb. It is well known that Hb has a close affinity on its scavenger protein haptoglobin (Hp) and hemopexin (Hx) to form the Hb/Hp/Hx complex. We therefore consider this process is hold the main effect on the change of HDL’s inflammatory properties in SCD patients and animals. We reasonably hope to study on the the association of Hb with HDL with the goal of finding the target for treatment in future.MethodFirst section:All the sample were from Wisconsin Sickle Cell Disease Comprehensive Center in Milwaukee for the study on p-HDL measurement by AE-HPLC assay. Total cholesterol (TC), triglyceride (TG) and cf Hb were determined by chemical cholormetric assay through enzyme-catalytic reaction. We took Dextran-MgCl2and the newly-designed AE-HPLC assay to isolate HDL separately. The influence on HDL oxidation by Hb and XO can be determined by spectrophotometry. To testify the advantage of HDL isolation by AE-HPLC assay, we analyze the proteins separated by HPLC by Immunoblot. To further determine p-HDL level in plasma, we install dual post-column reactor (PCR) to follow AE-column. Cu2+and DCFH were pumped into liquid to associate with HDL to induce oxidative reaction. P-HDL peak was detected by the fluorescence detector (FLD) and the p-HDL data were obtained through correct calculation. And6plasma sample from respective SCD patients and healthy donors were used to analyze p-HDL level determined by the HPLC system. Furthermore, we do p- HDL data analysis by linear regression analysis and other statistic assay to compare with the two assays on HDL-isolation and p-HDL measurement.Secend section:32plasma sample from SCD patients and28plasma sample from healthy donors were used for this study. For animal research we used WT、Hp-/-、Hx-/-SCD/non-SCD mice(n=8) respectively. ELISA or Immunoprecipitation (IP)+Immunoblot assay were used to measure ApoA-1and Hb/Hp/Hx in plasma or HDL. Chemical cholormetric assay was used for lipid and heme、IL-6、IL-10and other inflammatory factors determination. ROS and RNS were detected by respective fluorescence probe. We took the Bio-layer Interferometry (BLI) Octet System to analysis the interactions among Hb, Hp, Hx and ApoA-1molecules. We determined p-HDL level in plasma by AE-HPLC assay mentioned by the frist section. We obtained HDL Inflammatory Index (HII) by Monocyte Chemotaxis assay (MCA) to know the inflammatory properties of HDL among groups. On the basis of these, we administrated SCD mice with ApoA-1mimetic peptides D-4F (10μg/d,4weeks) for treatment, and measured the HDL-associated Hb/Hp/Hx, HII and the ability to bind with Hb by HDL and ApoA-1particles by Octet.ResultFirst section:Plasma TC was decreased in SCD patients, but TG was not different. Our data shown p-HDL level was positively correlated with cf Hb concentration in plasma. And cf Hb inside sickle cells is released into blood caused by hemolysis. It is also the main process during SCD. So cf Hb is the main factor to increase p-HDL level in plasma. Cf Hb and XO can accelerate the rate of HDL oxidation measured by UV-Spectrometer. Meanwhile, we found in HPLC with the previously reported method, the albumin peak is partly overlapped over the HDL peak to interfere the analysis. Thus we seek to remove cf Hb, XO and albumin to avoid their influence on oxidative rate. In our study, we demonstrated that cf Hb, albumin and XO can be removed step by step to purify HDL through AE-HPLC assay with principle of Anion exchange column’s binding affinity on different anion in proteins. Accordingly, we improved the "method" in HPLC based on previous report. HDL can be isolated under18%Solvent B without interference of cf Hb, albumin and XO. And we found p-HDL level from HPLC assay is quite lower than that from the plate assay. It suggests that XO (with xanthine) and cf Hb can accelerate HDL oxidation and increase p-HDL level. Thus AE-HPLC-PCR assay can effectively reduce the oxidant unavoidable in plate assay to improve the accuracy and detect the influence on p-HDL resulted by SCD.Secend section:In our study, we also found the elevated proinflammatory factors heme, ROS, IL-6, IL-10as well as less PON1and other anti-inflammatory proteins. It shown us there was chronic persistent inflammatory response in plasma of SCD and the production of p-HDL.In our study, we found less TC and ApoA-1, but higher Hb, Hp and Hx levels in ApoA-1contained in HDL cargoes of SCD by Sandwich ELISA, compared with control. We further confirmed that ApoA-1-associated Hb/Hp/Hx was positively relevant with HDL inflammatory index and LOOH that may reflect inflammatory response only in body of SCD group. It has been further proved that much more Hb, Hp and Hx than those of control specificly bound with ApoA-1particles in HDL by immunoprecipitation (IP) and immunoblot. HDL associated Hb/Hp/Hx through ApoA-I particles inside, which was well demonstrated by Bio-layer Interferometry (BLI) Octet System. Data from monocyte chemotaxis assay (MCA) shown that SCD did not change originally ant-inflammatory properties of HDL in Hp-/-mice, MCA<1. Associated Hb in HDL was even lower in Hp-/-mice of SCD than that of other SCD mice. HPLC isolated lipoproteins and shown lower p-HDL level in Hp-/-mice. We treat SCD mice with ApoA-1mimetic peptides, D-4F. D-4F can reduce Hb/Hp/Hx associated with HDL and make HDL return to become anti-inflammatory (MCA<1) in SCD mice.Conclusion1. We firstly and successfully separate oxidative factors to purify HDL with AE-HPLC assay and establish a more accurate assay to determine p-HDL level. It is applicable to assess carodiovascular risk in clinic job in future.2. We further seek to explore the mechanism on the generated proinflammatory properties of HDL and demonstrated that Hb-Hp is the critical passway to make HDL become proinflammatory.3.D-4F can be applied to reverse p-HDL to anti-inflammation. It suggests that the depolymerization of Hb/Hp/Hx may be a new target for treatment. It is also meaningful to treat dyslipidemia and the related angiopathy. |
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