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.

Differential Gene Expression by RNA-Seq Analysis of the Primo Vessel in the Rabbit Lymph.

For the connectome of primo vascular system, some long-type primo vessels dyed with Alcian blue injected into inguinal nodes, abdominal node, and axially nodes were visualized, which passed over around the vena cava of the rabbit. The Alcian blue dye revealed primo vessels and colored blue in the rabbit lymph vessels. The length of long-type primo vessels was 18 cm on average, of which diameters were about 20-30 µm, and the lymph vessels had diameters of 100-150 µm. Three different tissues of pure primo vessel, mixed primo + lymph vessel, and only lymph vessel were made to undergo RNA-Seq analysis by next-generation sequencing. We also analyzed differentially expressed genes (DEGs) from the RNA-Seq data, in which 30 genes of the primo vessels, primo + lymph vessels, and lymph vessels were selected for primo marker candidates. From the plot of DEG analysis, 10 genes had remarkably different expression pattern on the Group 1 (primo vessel) vs Group 3 (lymph vessel). With Fragments Per Kilobase of exon per Million the cutoff p-value for each gene was < 0.05. Fragments Per Kilobase of exon per Million of the 10 genes such as IGHM, HLA-DRA, HIST1H41, LPL, CD36, SRGN, DGAT2, SNCG, CD48, and GPD1 for primo vessels compared with those of lymph vessels increased twice or thrice. These results suggest that the selected genes could be used for the specific marker to construct primo connectome of circuit system in the rabbit.

Primo Vessel Stressed by Lipopolysaccharide in Rabbits.

For tracking the primo vascular system, we observed the primo vessels in vivo in situ using the lipopolysaccharide (LPS) response in the lymphatic vessels of a rabbit. Injection of LPS (200 µg/kg) into the lymph nodes resulted in greatly stained primo vessels, which were swollen in some cases. We were able to obtain comparative images through alcian blue and diaminobenzidine staining, which clearly showed different morphologies of the primo vessels. The mechanism causing the response of the primo vessels to the injected LPS is still unclear; however, these results might be a first attempt at giving an explanation of the function of the primo vascular system and identifying the changes in the structure and function of the primo vascular system in response to an external stimulus such as an injection of LPS.

Effective isolation of primo vessels in lymph using sound- and ultrasonic-wave stimulation.

The effects of stimulation with sound and ultrasonic waves of a specific bandwidth on the microdissection of primo vessels in lymphatic vessels of rabbit were investigated. The primo vessels stained with alcian-blue dye injected in the lymph nodes were definitely visualized and more easily isolated by sound-wave vibration and ultrasonic stimulation applied to rabbits at various frequencies and intensities. With sound wave at 7 Hz and ultrasonic waves at 2 MHz, the probability of detecting the primo vessels was improved to 90%; however, without wave stimulation the probability of discovering primo vessels was about 50% only. Sound and ultrasonic waves at specific frequency bands should be effective for microdissection of the primo vessels in the abdominal lymph of rabbit. We suggest that oscillation of the primo vessels by sound and ultrasonic waves may be useful to visualize specific primo structure, and wave vibration can be a very supportive process for observation and isolation of the primo vessels of rabbits.

Observation of a long primo vessel in a lymph vessel from the inguinal node of a rabbit.

Though primo vessels are frequently found in the lymph near the abdominal aorta of rabbit by Alcian blue dye, the reproductions are still difficult to require considerable skills and technical know-how at dissected tissue of animal species. However, in the inguinal lymph node of a rabbit we found a long-type primo vascular system (LTP) dyed with Alcian blue, from an abdominal lymph vessel to an inguinal lymph node. The length of LTP was over an average length of 9.1 cm. The average diameters of the primo and the lymph vessels were about 23.9 µ m and 242 µ m, respectively. The primo vessels were not floating but adhered to lymph vessels with fascial connective tissue. These primo vessels might be a functional integration in the lymph system.

Loss of caspase-1 gene expression in human gastric carcinomas and cell lines.

The caspases are a family of aspartic acid-specific proteases that fulfill varied and often critical roles in mammalian apoptosis or in the proteolytic activation of cytokines. Caspase-1 (interleukin-1beta-converting enzyme) is a member of the cysteine protease family, which cleaves target proteins following aspartic acid residues. We investigated caspase-1 expression in stomach cancer, tissues and cell lines. Of 301 consecutive gastric carcinomas, 58 cases (19.3%) showed the expressional loss of caspase-1. Loss of caspase-1 expression was significantly associated with pTNM stage (p=0.03), lymph node metastasis (p=0.01) and patient survival (p<0.01). Caspase-1 expression was also significantly correlated in an inverse manner with p53 expression (p<0.01). Among the 11 gastric cancer cell lines examined, three cell lines showed loss of expression at the protein and mRNA levels. On treatment with 5-aza-2'-deoxycytidine (5-aza-C), and/or trichostatin A (TSA), all three cell lines re-expressed caspase-1 mRNA. The above findings suggest that epigenetic events such as DNA methylation and histone deacetylation play important roles in the regulation of caspase-1, and that loss of caspase-1 expression is associated with poor survival in gastric carcinoma.