Nathan Abel Arul, Dixit Madhulika, Babu Subash, Balakrishnan Anand Setty
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India.
Laboratory of Vascular Biology, Department of Biotechnology, Bhupat Joyti Metha School of Biosciences and Bioengineering, Indian Institute of Technology Madras, Chennai, India.
Trop Med Int Health. 2017 Nov;22(11):1414-1427. doi: 10.1111/tmi.12969. Epub 2017 Sep 20.
The underlying problem in lymphatic filariasis is irreversible swelling of the limbs (lymphoedema), which is a unique feature of lymphatic insufficiency. It is still unclear whether the natural ability of lymphatics to form functional lymphatic vasculature is achieved or attenuated in the lymphoedemal pathology. Clinical studies have clearly shown that circulating lymphatic progenitors (CLPs), a subset of bone marrow-derived mononuclear cells (PBMCs), contribute to post-natal lymph vasculogenesis. CLP-based revascularisation could be a promising strategy to bypass the endothelial disruption and damage incurred by the filarial parasites. Thus our aim was to compare and characterise the functional prowess of PBMCs in physiological and lymphoedemal pathology.
PBMCs were isolated from venous blood sample from drug-naive endemic normals (EN) and drug-deprived filarial lymphoedema (FL) individuals using density gradient centrifugation. Adhesion, transwell migration and in vitro matrigel assays were employed to characterise the lymphvasculogenic potential of PBMCs. CLPs were phenotypically characterised using flow cytometry; expression levels of lymphatic markers and inflammatory cytokines were quantified using qRT-PCR and ELISA, respectively.
PBMCs from FL group display poor adherence to fibronectin (P = 0.040), reduced migration towards SDF-1α (P = 0.035), impaired tubular network (P = 0.004) and branching point (P = 0.048) formation. The PBMC mRNA expression of VEGFR3 (P = 0.039) and podoplanin (P = 0.050) was elevated, whereas integrin α9 (P = 0.046) was inhibited in FL individuals; additionally, the surface expression of CD34 (P = 0.048) was significantly reduced in the FL group compared to the EN group.
PBMCs from filarial lymphoedema show defective and dysregulated lymphvasculogenic function compared to endemic normals.
淋巴丝虫病的根本问题是肢体不可逆肿胀（淋巴水肿），这是淋巴功能不全的一个独特特征。目前尚不清楚在淋巴水肿病理过程中，淋巴管形成功能性淋巴脉管系统的天然能力是得以实现还是减弱。临床研究已明确表明，循环淋巴祖细胞（CLP），即骨髓来源的单核细胞（PBMC）的一个亚群，有助于出生后淋巴脉管生成。基于CLP的血管再生可能是一种有前景的策略，可绕过丝虫寄生虫引起的内皮破坏和损伤。因此，我们的目的是比较并表征PBMC在生理和淋巴水肿病理状态下的功能能力。
使用密度梯度离心法从未经治疗的地方性正常人群（EN）和未经药物治疗的丝虫性淋巴水肿（FL）个体的静脉血样本中分离PBMC。采用黏附、Transwell迁移和体外基质胶试验来表征PBMC的淋巴脉管生成潜力。使用流式细胞术对CLP进行表型特征分析；分别使用qRT-PCR和ELISA对淋巴标志物和炎性细胞因子的表达水平进行定量。
FL组的PBMC对纤连蛋白的黏附性较差（P = 0. ***），向SDF-1α的迁移能力降低（P = 0. ***），管状网络形成受损（P = 0. ***），分支点形成受损（P = 0. ***）。FL个体中VEGFR3（P = 0. ***）和血小板内皮细胞黏附分子（P = 0. ***）的PBMC mRNA表达升高，而整合素α9（P = 0. ***）受到抑制；此外，与EN组相比，FL组中CD34的表面表达显著降低（P = 0. ***）。
与地方性正常人群相比，丝虫性淋巴水肿患者的PBMC表现出有缺陷且失调的淋巴脉管生成功能。
