Analysis and Differential Expression of Primo Genes Using RNA-Seq and qRT-PCR Experiments.

Although the existence of the primo vasculature system has been shown in many species, including mice, rats, rabbits and humans, the biological role of this system, including expression of genes and proteins, has not yet been investigated. Especially the transcriptional action by mRNA, which is required for biological action, needs to be studied in primo vasculature biology. Differentially expressed genes in both isolated primo vessels and lymphatic vessels of rabbits were analyzed by RNA sequencing experiments. Primer efficiency and RNA purity of the primo vessels under lipopolysaccharides were confirmed prior to performing real-time qRT-PCR analysis following RNA extraction. We demonstrated that FLT4 was enriched in primo vessels and that several genes, including HSPH1 and EPHB2, were highly expressed in primo vessels compared with lymphatic vessels. Our data show that almost all genes, except HSPA4, were increased or sustained in isolated primo vessels compared with lymphatic vessels (FLT4 2.58 fold, HSPH1 1.83 fold, EPHB2 1.52 fold; whereas HSPA4 decreased 0.50 fold), suggesting primo vessels as a central regulator in diverse physiology. This implies that FLT4, HSPH1, and EPHB2 in high amounts may be involved in the functional activity of primo vessels. Our experimental data show that several genes are highly enriched in primo vessels in the lymphatic vessels of the rabbit.

Expression of Genes in Primo Vasculature Floating in Lymphatic Endothelium Under Lipopolysaccharide and Acupuncture Electric Stimulation.

It is known that the primo vascular system (PVS) includes the primo nodes and vessels. However, the relevant genes in the PVS system for both pathologic and physiologic condition are poorly understood. Here, we first examined the gene expression in primo vessels (PVs) floating in lymphatic endothelium by isolation of PVS and lymphatic vessels (LVs) containing PVS. To investigate therapeutic effects, both PVs and LVs containing PVS were isolated after lipopolysaccharide injection and acupuncture electric stimulation at two acupoints Joksamni (ST36) and Hapgok (LI04) following lipopolysaccharide injection. We used reverse transcriptase-polymerase chain reaction to examine expression of lymphatic endothelial cell markers and inflammatory related genes. We found that lymphatic endothelial cell markers such as fms-related tyrosine kinase 4 (Flt4), lymphatic vessel endothelial receptor (Lyve-1), prospero homeobox protein 1 (Prox-1), and podoplanin (Pdpn) were highly expressed in PV compared to that of lymphatic endothelium, suggesting pivotal roles of PV in LV under inflammation. Furthermore, lymphatic-related genes including metal-response element-binding transcription factor 2 (Mtf2), hypoxia inducible factor (Hif1a), angiotensin II type 1 receptor (Agtr1), and angiotensin II type 2 receptor (Agtr2) were also overall increased in PV, and remarkably increased and these genes except peroxisome proliferator-activated receptor gamma (Pparg) after acupuncture electric stimulation in two acupoints implying central role of PV by gene activation.