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.

Injection Effect of Anti-CD3 Monoclonal Antibody on Primo Vessel in Lymph Vessel of Rabbit with Lipopolysaccharide-Induced Inflammation.

Background: The primo vascular system can be viewed as a circulatory system that plays a therapeutic function in regenerating the body tissue. The anti-CD3 monoclonal antibody was used as an immunotherapeutic agent to treat the novel coronavirus infection (COVID-19). Objectives: In this study, we observed the effect of injecting lymph nodes with Foralumab, an anti- human CD3 epsilon therapeutic monoclonal antibody, on primo vessels. Methods: The structure and atomic stoichiometry of the antibody were determined by transmission electron microscopy and energy dispersive spectroscopy. Alcian blue dying solution was injected into the lymph nodes of the abdominal vena cava of rabbits, and the solution further flowed into the lymph vessels. Results: A primo vessel with primo nodes stained with Alcian blue was clearly visible in the lymph vessel. By injecting Foralumab into lymph nodes of rabbits with lipopolysaccharide-induced inflammation, the floating primo vessel in the lymph vessel appeared thicker and was distinctly visible. Conclusion: The observation of the primo vessel post-treated with Foralumab in the inflamed lymphatic system suggests the possibility of a functional role of the primo vascular circulatory system in pathophysiological conditions.

Primo Bundles Identified by Microcomputed Tomography in Primo Vascular Tissue on the Surface of Rat Abdominal Organs.

BACKGROUND: The primo vascular system (PVS) is a novel network composed of primo nodes (PNs) and primo vessels (PVs). Currently, its anatomy is not fully understood. OBJECTIVES: The aim of this study was to elucidate the three-dimensional PN-PV structure. METHODS: Organ-surface PVS tissue was isolated from healthy and anemic rats. The tissues were analyzed by X-ray microcomputed tomography (CT), hematoxylin and eosin staining, and scanning electron microscopy. RESULTS: From CT images, we identified one or more bundles in a PV. In the PN, the bundles were enlarged and existed in isolation and/or in anastomosis. The transverse CT images revealed four areas of distinct intensities: zero, low, intermediate, and high. The first two were considered to be the sinuses and the subvessels of the PVS and were identified in the hematoxylin and eosin-stained PN sections. The enlargement of the PN from anemic rats was associated with an increase in the intermediate-intensity area. The high-intensity area demarcated the bundle and was overlapped with the mesothelial cells. In scanning electron microscopy, the PV bundles branched out, tapering down to a single bundle at some distance from the PN. Each bundle was composed of several subvessels (∼5 µm). Clustered round microcells (1-25 µm), scattered flat oval cells (∼15 µm), and amorphous extracellular matrix were observed on the surface of the PVS tissue. CONCLUSIONS: The results newly showed that the primo bundle is a structural unit of both PVs and PNs. A bundle was demarcated by high CT intensity and mesothelial cells and consisted of multiple subvessels. The PN bundles contained also sinuses.

Mesothelial Cells Covering the Surface of Primo Vascular System Tissue.

The primo vascular system (PVS) is reported to have a periductium composed of cells with spherical or spindle-shaped nuclei and abundant cytoplasm. However, little is known about these periductium cells. In this study, we examined the morphological features of cells covering the PVS tissue isolated from the surface of abdominal organs of rats. By hematoxylin and eosin (H&E) staining, we observed a layer of dark nuclei on the basement membrane at the borders of the sections of primo node (PN), primo vessel (PV), and their subunits. The nuclei appeared thin and linear (10-14 µm), elliptical (8-10 × 3-4 µm), and round (5-7 µm). The borders of the PVS tissue sections were immunostained with a selective antibody for mesothelial cells (MCs). Areas of immunoreactivity overlapped with the flattened cells are shown by hematoxylin and eosin staining. By scanning electron microscopy, we further identified elliptical (11 × 21 µm) and rectangular squamous MCs (length, 10 µm). There were numerous stomata (∼200 nm) and microparticles (20-200 nm) on the surface of the PVS MCs. In conclusion, this study presents the novel finding that the PVS periductium is composed of squamous MCs. These cells tightly line the luminal surface of the PVS tissue, including PNs, PVs, and small branches of the PVs in the abdominal cavity. These results will help us to understand the physiological roles such as hyaluronan secretion and the fine structure of PVS tissue.

Putative Primo-Vascular System in Rabbit Placenta.

The primo vascular system (PVS) is a very important topic of study nowadays because of their role in transport and regeneration of tissue and in cell migration and cancer metastasis. The PVS was detected in different organs of the rabbit but not in the placenta. In this work, we observe the PVS inside the blood vessels of the placenta for the first time. The main characteristic features of the primo vessels (PVs) from the rabbit placenta were in agreement with the PVS in different organs of animals, including the rod-shaped nuclei and their arrangement.

Evidence for novel tubular-bundle structures entangled in the fascia of the inner abdominal wall of a rat.

After incubation with Janus Green B in the peritoneal cavities of rats, lymph-vessel-like structures (LSs) were noticed under a stereomicroscope to run parallel to each other with rectangular branches on the inner abdominal wall; rarely were these LSs seen to connect with peritoneal LSs. These LSs were identified by using fluorescence microscopy (FM) at a magnification of 1,000X and confocal laser scanning microscopy (CLSM) to be lymph-vessel-like bundle structures (LBSs). Serial cross-sections of these LBSs were microscopically examined by using hematoxylin and eosin staining, Mattson trichrome staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry (IHC) with Lyve 1 and CD 31. The histology data from these LBSs revealed such novel characteristics as parallel clusters of live cells wrapped by collagen fibers of the fascia and an IHC different from those of lymphatic and blood vessels, being the novel bundle structures (NBSs). Under FM and CLSM with optical sections, some of the NBSs were observed to be able to swell like tiny balloons, implying that the bundle structures were hollow. Moreover, transmission electron microscopy images of two different cross-sections of an NBS showed it to be composed of four parallel tubules involving three kinds of sinuses with neither axons nor Schwann cells in the outermost wall, thus being a novel tubular-bundle structure (NTBS). The results of this research make evident with high repeatability (10/11) that beyond the orthodoxy of a single-tube system of blood and lymph vessels, a system of NTBSs is widely entangled in the fascia of the inner abdominal wall of a rat. Thus, the author suggests that NTBS-related functions and the entire NTBS network should be explored.

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.

Various stem cells in acupuncture meridians and points and their putative roles.

Traditional Chinese and Korean medicine uses various manipulations on acupuncture points/acupoints that are located along imaginary lines on the surface of a human body, which are called 'meridians'. Acupuncture has been used from the ancient time till now to cure various diseases, including for the purpose of regenerative medicine. In various studies, meridians are alternatively called as Bong-Han ducts, primo vessels, or hyaluronic-acid rich ducts, while acupoints are called Bong-Han corpuscles, primo nodes, or hyaluronic-acid rich nodes. Meridians and acupuncture points form a system that is now called primo vascular system (PVS), which is claimed to contain various kinds of stem cells. The stem cell size is between 1-5 microns. The smallest is the primo microcells that have a putative role in regeneration. Other stem cells are adult pluripotent and hematopoietic stem cells that play a role in extra bone marrow hematopoiesis. The presence of PVS has been reproduced by many studies. However, the various stem cells need further studies to prove their existence and function, and harvesting PVS to isolate the stem cells might harm the health of the donor.

Protocol for Detecting the Primo Vascular System in the Lymph Ducts of Mice.

The primo vascular system (PVS), which is the proposed conduit for the acupuncture Qi, is a complex network distributed throughout an animal's body. However, even with a microscope, it is not easily detectable because of its transparency. Thus, its existence is largely unknown in current anatomy. A convincing demonstration of its existence is needed. The lymph-primo vessel (PV), which is a subsystem of the PVS, is a very effective visual demonstration of the PVS. The lymph-PVS is a mobile threadlike structure floating in lymph ducts that has been observed in rabbits, rats, and mice by several independent teams. The involved techniques are novel and rather complicated; therefore, we have already provided detailed protocols for the surgery; for the injection of the staining dye; and for the detection, extraction, and identification of the PVS in rabbits and rats. However, the mouse is one of the most important laboratory animals used for various biomedical research purposes. For the convenience of researchers who wish to initiate the PVS experiments in mice, we provide a shortened version of the protocol, despite many similarities with previously published protocols. Thus, researcher can easily obtain the samples of the lymph-PVS of mice.

A Novel Technique for Visualizing the Intralymphatic Primo Vascular System by Using Hollow Gold Nanospheres.

Until recently, the primo vascular system (PVS) has been unnoticed by most anatomists due to the small diameter and translucent features of the threadlike network. These properties make primo vessels (PVs) difficult to visualize for harvest and for further characterization. One particular PVS subtype that is located within the lymphatic vessels (LVs) is of strong interest because with a proper contrast, these long PVs can be visualized through the transparent LV wall and can be harvested to provide sufficient sample material for analysis. The most common method to visualize this PVS subtype utilizes Alcian blue as the contrast agent. This technique is effective, but tedious, and has relatively low repeatability. The purpose of this study was to develop a new technique that allows reliable visualization of the intralymphatic PVS (IL-PVS) in a user-friendly manner. The method was designed to provide optical contrast to the PVS by taking advantage of the porous nature of the PV's external wall and interstitial matrix. Turquoise-green-colored hollow gold nanospheres (HGNs) in the size range of 50-125 nm were found to provide excellent optical contrast for the IL-PVS in rats. The PVS was visualized within 10 minutes after HGN administration at a 95% success rate.

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.

Motion properties of the sanals of the primo vascular system under a magnetic field.

The motion properties of the sanals of the primo vascular system were investigated under a low static magnetic field of 100 Oe. Sanals of about 1 µm were selected and separated from the primo vessels and nodes on a rabbit's organ surface. The average velocity of five sanals in a physiologic saline solution parallel and perpendicular to the direction of the applied magnetic field was approximately 1.0 pixel/second in random directions, which implies that the rotating motion of sanals with nuclei composed of DNA containing many inorganic magnetic elements such as manganese and cobalt is monotonically weakened by increasing an applied magnetic field.