Pericytes in Cutaneous Wound Healing.

Most of the studies on cutaneous wound healing are focused on epidermal closure. This is obviously important, as the epidermis constitutes the main barrier that separates the inner organism from the environment. However, dermal remodeling is key to achieve long-lasting healing of the area that was originally wounded. In this chapter, we summarize what is known on the stromal components that strongly influence the outcome of healing and postulate that dedifferentiation of stably differentiated cells plays a major role in the initial response to wounding, as well as in long-term wound remodeling. Specifically, we explore the available evidence implicating skin pericytes, endothelial cells, Schwann cells, and macrophages as major players in a complex symphony of cellular plasticity and signaling events whose balance will promote healing (by tissue regeneration or repair) or fibrosis.

Pathological Process of Prompt Connection between Host and Donor Tissue Vasculature Causing Rapid Perfusion of the Engineered Donor Tissue after Transplantation.

The shortage of donors for transplantation therapy is a serious issue worldwide. Tissue engineering is considered a potential solution to this problem. Connection and perfusion in engineered tissues after transplantation is vital for the survival of the transplanted tissue, especially for tissues requiring blood perfusion to receive nutrients, such as the heart. A myocardial cell sheet containing an endothelial cell network structure was fabricated in vitro using cell sheet technology. Transplantation of the three-dimensional (3D) tissue by layering myocardial sheets could ameliorate ischemic heart disease in a rat model. The endothelial cell network in the 3D tissue was able to rapidly connect to host vasculature and begin perfusion within 24 h after transplantation. In this review, we compare and discuss the engineered tissue⁻host vasculature connection process between tissue engineered constructs with hydrogels and cell sheets by histological analysis. This review provides information that may be useful for further improvements of in vivo engineered tissue vascularization techniques.

A simple, one-step polychromatic staining method for epoxy-embedded semithin tissue sections.

Although conventional toluidine blue staining is a common technique used for rapid observation of semithin sections prior to transmission electron microscopy, it is monochromatic and insufficient for accurate identification of different tissue components by light microscopy. Additionally, polychromatic staining methods generally require step-by-step processes involving different dyes, and it is often difficult to balance the color tone of each step. In this study, we developed a simple polychromatic staining method for epoxy-embedded tissue sections. We stained preheated sections with an aqueous ethanol solution of azure B and basic fuchsin, with the addition of sodium tetraborate to enhance the staining efficacy. We optimized various staining conditions to enable sufficient coloration easily and consistently in a single, rapid staining step, using a single staining-mixture solution. Our method enabled clear differentiation of various tissue structures according to color tone and stain intensity, thereby facilitating the detection of fine structural differences, including various organelle and inclusion bodies. This technique represents a simple polychrome-staining method to allow more informative and convincing histological investigation in various fields of research and education.

Lactic Acid Bacteria Isolated from Japanese Fermented Fish (Funa-Sushi) Inhibit Mesangial Proliferative Glomerulonephritis by Alcohol Intake with Stress.

The aim of this study was to examine the effect of heat-killed Lactobacillus paracasei NFRI 7415 on kidney and bone in mice fed an ethanol-containing diet with stress. Eight-week-old Cril : CD1 mice were fed a control diet (CD), an alcohol diet (AD) (35.8% of total energy from ethanol), or an alcohol diet containing 20% heat-killed Lb. paracasei NFRI 7415 (107 CFU/g) (LD) for 4 weeks. Mice in the AD and LD groups also underwent restraint stress for two weeks from 13 days. The mice were placed in a 50 mL plastic tube, which had a small hole drilled around its base to allow ventilation, and restrained for 1 h every day. High final body weight was in the following order: CD, LD, and AD (p < 0.05). The heat-killed Lb. paracasei NFRI 7415 lowered liver total cholesterol concentration and plasma glutamic-oxaloacetic transaminase (GOT) level. In addition, fecal bile acids of the LD group were higher than in the AD group (p < 0.05). The glomerulus of the kidney in the AD group was observed to be more fibrotic than in the CD and LD groups with azan stain. Immunostaining confirmed that brown areas indicating the existence of mesangial cells were increased in the AD group, but not in the CD and LD groups. These results indicated that the heat-killed Lb. paracasei NFRI 7415 inhibited mesangial proliferative glomerulonephritis by alcohol intake with stress.

Morphological characterization of pulmonary microvascular disease in bronchopulmonary dysplasia caused by hyperoxia in newborn mice.

PURPOSE: Pulmonary microvascular injury is associated with the pathogenesis of bronchopulmonary dysplasia (BPD). To characterize the mechanisms of pulmonary vascular disease resulting from BPD, we studied the ultrastructural changes affecting pulmonary microvasculature. METHODS: Newborn ICR mice were exposed to 85% hyperoxia or normoxia for 14 days, and then normal air replacement conditions for the following 7 days. At postnatal day (P)14 and P21, lungs were harvested for ultrastructural examination and assessment of pulmonary hypertension. RESULTS: The ultrastructure of pulmonary microvasculature in the hyperoxia-exposed lungs revealed a collapsed capillary lumen. This was due to the abnormal morphology of endothelial cells (ECs) characterized by heterogeneously thick cytoplasm. Compared to normal air controls, the specimens displayed also remarkably thick blood-air barriers (BABs), most of which were occupied by EC layer components. Structural changes were accompanied by increased pulmonary artery medial thickness and right ventricular hypertrophy (RVH). Moreover, abnormalities in ECs persisted even after exposure to 7 days of normal air replacement conditions. Results were confirmed by morphometric quantification. CONCLUSION: Our results suggest that the abnormal morphology of capillary ECs and thick BABs correlates with pulmonary artery remodeling and RVH. These ultrastructural changes might represent possible mechanisms of secondary pulmonary hypertension in BPD.

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Foxc2 influences alveolar epithelial cell differentiation during lung development.

FOXC2, a forkhead transcriptional factor, is a candidate gene for congenital heart diseases and lymphedema-distichiasis syndrome and yellow nail syndrome; however, there are no reports on Foxc2 and the development of the lung. We have identified lung abnormalities in Foxc2-knockout embryos during investigation of cardiac development. The aim of this study was to clarify the morphological characteristics during lung development using ICR-Foxc2 knockout lungs. Mutant fetuses at embryonic days 10.5-18.5 were obtained from mating of Foxc2+/- mice and then analyzed. Notably, Foxc2-knockout lungs appeared parenchymatous and much smaller than those of the wild-type littermates. In the Foxc2 knockout lungs, the capillary beds remained distant from the alveolar epithelium until the late stages, the number of type2 alveolar cells per alveolar progenitor cell was lower and the type1 alveolar cells were thicker in Foxc2 knockout mice. In contrast, Foxc2 expression was only detected in the mesenchyme of the lung buds at E10.5, and it disappeared at E11.5 in Foxc2-LacZ knockin mice. Furthermore, the expression of Lef1 was significantly inhibited in E11.5 lungs. All of these results suggest that the abnormalities in Foxc2 knockout mice may involve maldifferentiation of alveolar epithelial cells and capillary vessel endothelial-alveolar epithelial approach as well as lymph vessel malformation. This is the first report about relationship between Foxc2 and lung development. This animal model might provide an important clue for elucidating the mechanism of lung development and the cause of respiratory diseases.

Pericytes promote abnormal tumor angiogenesis in a rat RG2 glioma model.

In glioma angiogenesis, tumor vessels cause morphological and functional abnormalities associated with malignancy and tumor progression. We hypothesized that certain structural changes or scantiness of functional pericytes may be involved in the formation of dysfunctional blood vessels in gliomas. In this study, we performed morphological examinations to elucidate the possible involvement of pericytes in brain tumor vessel abnormalities using a rat RG2 glioma model. After implantation of RG2 glioma cells in the syngeneic rat brain, gliomas were formed as early as day 7. In immunohistochemical examinations, desmin-positive pericytes, characterized by morphological abnormalities, were abundantly found on leaky vessels, as assessed by extravasation of lectin and high-molecular-weight dextrans. Interestingly, desmin-positive pericytes seemed to be characteristic of gliomas in rats. These pericytes were also found to express heat-shock protein 47, which plays an important role in the formation of the basement membrane, suggesting that RG2 pericytes promoted angiogenesis by producing basement membrane as a scaffold for newly forming blood vessels and caused functional abnormalities. We concluded that RG2 pericytes may be responsible for abnormal tumor angiogenesis lacking the functional ability to maintain the blood-brain barrier.

Xenogeneic transplantation of human adipose-derived stem cell sheets accelerate angiogenesis and the healing of skin wounds in a Zucker Diabetic Fatty rat model of obese diabetes.

INTRODUCTION: Diabetic patients with foot ulcers often suffer impaired wound healing due to diabetic neuropathy and blood flow disturbances. Direct injection of human adipose-derived stem cells (hASCs) effectively accelerates wound healing, although hASCs are relatively unstable. METHODS: We developed an optimized protocol to engineer hASC sheets using temperature-responsive culture dishes to enhance the function and stability of transplanted cells used for regenerative medicine. Here, we evaluated the efficacy of hASC sheets for enhancing wound healing. For this purpose, we used a xenogeneic model of obese type 2 diabetes, the Zucker Diabetic Fatty rat (ZDF rat), which displays full-thickness skin defects. We isolated hASCs from five donors, created hASC sheets, and transplanted the hASC sheets along with artificial skin into full-thickness, large skin defects (15-mm diameter) of ZDF rats. RESULTS: The hASC sheets secreted angiogenic growth factors. Transplantation of the hASC sheets combined with artificial skin increased blood vessel density and dermal thickness, thus accelerating wound healing compared with that in the controls. Immunohistochemical analysis revealed significantly more frequent neovascularization in xenografted rats of the transplantation group, and the transplanted hASCs were localized to the periphery of new blood vessels. CONCLUSION: This xenograft model may contribute to the use of human cell tissue-based products (hCTPs) and the identification of factors produced by hCTPs that accelerate wound healing.

Electron microscopic study of capillary network remodeling in the extensor digitorum longus muscle of normal adult rat.

Capillary networks demonstrate structural changes during maturation, aging, vascular disease, and cancer. Their morphological structure and function have an important influence on each other. Understanding the process of morphological vascular changes in the capillary network with advancing age may help overcome fatal vascular diseases. Aging-related structural changes of the capillary segments may accompany degeneration and regeneration of muscle fibers and serve to remodel the capillary network as a means of adapting to the changing environment. However, difficulty in obtaining human samples has hampered clarification of these microstructural changes. Herein, we examined serial ultrathin sections of capillary segments in the extensor digitorum longus muscle of normal mature (12 months old) rats in an attempt to analyze their structural changes. After bifurcation, a minimum of one capillary segment was filled with erythrocytes and was found to have fenestrations and plural endothelial disruptions, or pores, at the fenestrated portions. Some of the stagnated erythrocytes demonstrated extended protrusions, and their processes appeared to penetrate the basal lamina through the pores. These findings can also show that capillary segments are involved in partial remodeling of the capillary network. A better understanding of age-related structural changes of the capillary networks will help in fine-tuning novel vascular therapy for not only several fatal vascular diseases but also malignant tumors.

Allogeneic Transplantation of an Adipose-Derived Stem Cell Sheet Combined With Artificial Skin Accelerates Wound Healing in a Rat Wound Model of Type 2 Diabetes and Obesity.

One of the most common complications of diabetes is diabetic foot ulcer. Diabetic ulcers do not heal easily due to diabetic neuropathy and reduced blood flow, and nonhealing ulcers may progress to gangrene, which necessitates amputation of the patient's foot. This study attempted to develop a new cell-based therapy for nonhealing diabetic ulcers using a full-thickness skin defect in a rat model of type 2 diabetes and obesity. Allogeneic adipose-derived stem cells (ASCs) were harvested from the inguinal fat of normal rats, and ASC sheets were created using cell sheet technology and transplanted into full-thickness skin defects in Zucker diabetic fatty rats. The results indicate that the transplantation of ASC sheets combined with artificial skin accelerated wound healing and vascularization, with significant differences observed 2 weeks after treatment. The ASC sheets secreted large amounts of several angiogenic growth factors in vitro, and transplanted ASCs were observed in perivascular regions and incorporated into the newly constructed vessel structures in vivo. These results suggest that ASC sheets accelerate wound healing both directly and indirectly in this diabetic wound-healing model. In conclusion, allogeneic ASC sheets exhibit potential as a new therapeutic strategy for the treatment of diabetic ulcers.

Vasohibin-2 modulates tumor onset in the gastrointestinal tract by normalizing tumor angiogenesis.

BACKGROUND: Vasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of ApcMin/+ mice and ApcMin/+/Vash2-/- mice. METHODS: ApcMin/+ mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. ApcMin/+ mice were mated with Vash2-/- mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2-/- gene by PCR. Intestinal tumors from ApcMin/+ mice and ApcMin/+/Vash2-/- mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed. RESULTS: We found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in ApcMin/+ mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2-/- mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2-/- mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine. CONCLUSION: We propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.

Morphological and functional characterization of non-alcoholic fatty liver disease induced by a methionine-choline-deficient diet in C57BL/6 mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), appears to be increasingly common worldwide. Its histopathology and the effects of nutrition on liver function have not been fully determined. AIM: To elucidate the cellular mechanisms of NAFLD induced by a methionine-choline-deficient (MCD) diet in mice. Particular focus was placed on the role of phagocytic cells. METHODS: Male C57BL/6 mice were fed an MCD diet for 30 weeks. A recovery model was also established wherein a normal control diet was provided for 2 weeks after a period of 8, 16, or 30 weeks. RESULTS: Mice fed the MCD diet for ≥ 2 weeks exhibited severe steatohepatitis with elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Steatohepatitis was accompanied by the infiltration of CD68-positive macrophages (Kupffer cells). The severity of steatohepatitis increased in the first 16 weeks but was seen to lessen by week 30. Fibrosis began to develop at 10 weeks and continued thereafter. Steatohepatitis and elevated serum hepatic enzyme concentrations returned to normal levels after switching the diet back to the control within the first 16 weeks, but fibrosis and CD68-positive macrophages remained. CONCLUSIONS: The histopathological changes and irreversible fibrosis seen in this model were caused by prolonged feeding of an MCD diet. These results were accompanied by changes in the activity of CD68-positive cells with temporary elevation of CCL-2, MMP-13, and MMP-9 levels, all of which may trigger early steatohepatitis and late fibrosis through phagocytosis-associated MMP induction.

Intravitreal injection of erythropoietin protects against retinal vascular regression at the early stage of diabetic retinopathy in streptozotocin-induced diabetic rats.

A single intravitreal injection of erythropoietin (EPO) (50 ng/eye) or phosphate-buffered saline was administered to 5-week-old Sprague-Dawley rats at the onset of diabetes mellitus (DM) to determine and evaluate the protective effect of EPO on retinal microvessels. DM was induced by an intraperitoneal injection of streptozotocin (STZ; 60 mg/kg body weight). Morphological changes in microvessels in flat retinal preparations were evaluated during the subsequent 4 weeks by three-dimensional imaging of all blood vessels stained with fluorescein isothiocyanate-conjugated tomato lectin, following immunofluorescence techniques. No marked differences were observed in the shape or density of retinal vessels and the number of retinal capillary branches of the four groups [control, EPO, DM, and DM/EPO] up to 4 weeks after STZ administration. We also observed unique type IV collagen-positive filamentous structures that lacked both cellular elements and blood circulation (lectin-/type IV+ acellular strands), suggesting regressed vessel remnants. The lectin-/type IV+ acellular strands were detected soon after the onset of DM in the diabetic rats, and the number of these structures increased in the DM group (P < 0.01). A single intravitreal injection of EPO caused a significant reduction in the number of lectin-/type IV+ acellular strands to levels observed in the control group. However, the lectin-/type IV+ acellular strands were observed in the central area of the retina near the optic disc in all four groups. Intravitreal injection of EPO resulted in downregulation of the EPO receptor, vascular endothelial growth factor (VEGF), and VEGF receptor at 4 weeks. We conclude that EPO may play a primary role against the progression of diabetic retinopathy by reducing blood vessel degeneration at a very early disease stage.

Serum alanine aminotransferase concentration as a predictive factor for the development or regression of fatty liver.

Serum alanine aminotransferase (ALT) concentration is the most commonly used marker for hepatocellular injury. We investigated the suitable cutoff value of serum ALT for the diagnosis or prediction of fatty liver. In 1578 Japanese adults (1208 men, 370 women; 35-69 years of age) who visited our center both in 2000 and between April 2007 and March 2008 (2007-2008), serum ALT concentration was an independent predictor of fatty liver in men in 2000 and in both sexes in 2007-2008. A significant increase in the frequency of fatty liver was detected in participants with elevated serum ALT concentrations, and serum levels of ALT in 2000 were associated with fatty liver in 2007-2008 when the cutoff value was set at 30 IU/L in men and 19 IU/L in women. The frequency of fatty liver in 2007-2008 was significantly lower in participants without fatty liver in 2000 whose serum ALT decreased between 2000 and 2007-2008. Our results suggest that serum ALT might be not only an indicator of fatty liver but also a predictor of the regression of fatty liver, and cutoff values of serum ALT of 30 IU/L in men and 19 IU/L in women are suitable for the screening of fatty liver.

Phenotypic changes and possible angiogenic roles of pericytes during wound healing in the mouse skin.

Pericytes (PCs) are attracting increasing attention as a crucial target for anti-angiogenic therapy. In this study, we sought to determine the functional significance of PCs during angiogenesis by using a skin wound healing model in which different angiogenic stages are identifiable. Angiogenesis was first observed on Day 3 after wounding and increased greatly on Day 5. On Day 5, the leading edge of the regenerating vessels (vascular advancing front; VAF) appeared to be composed of immature vessels, and was further divided into "tip" and "following" regions according to maturational differences. PCs distributed in regenerating vessels showed phenotypic differences according to different regions. PCs that expressed PDGFR-ß alone and lacked vascular basement membrane (BM) were predominant in the tip region of the VAF, while PCs that expressed both PDGFR-ß and NG2 with their BM coating were numerous in the following regions toward the rear of the VAF. Moreover, PCs in the VAF expressed VEGF-A and associated with most proliferating endothelial cells (ECs). VEGF-A expression of PCs and the proliferating ECs totally disappeared in the region toward the rear of the VAF. We conclude that PCs can differ in their phenotype according to the stage of angiogenesis during wound healing. They may promote angiogenesis at the initial stage but might in turn stabilize the newly formed vessels at the later stage.

4-Hydroxy-2-nonenal upregulates and phosphorylates cytosolic phospholipase A(2) in cultured Ra2 microglial cells via MAPK pathways.

Several studies have suggested the involvement of neuroinflammation in the pathomechanism of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We recently demonstrated increased levels of protein-bound 4-hydroxy-2-nonenal (HNE) as a highly reactive lipid peroxidation product and cytosolic phospholipase A(2) (cPLA(2)) as a proinflammatory enzyme in glial cells as well as motor neurons in the spinal cord of sporadic ALS patients. However, a link between HNE and cPLA(2) in ALS remains to be determined. To address this issue, we investigated effects of HNE stimulation on the state of cPLA(2) expression in cultured microglial cell line (Ra2). Exposure of Ra2 cells to HNE significantly increased expression levels of cPLA(2) and its activated form phosphorylated at amino acid residue S(505) (p-cPLA(2)) on immunoblots. Pretreatment of Ra2 cells with the antioxidant N-acetylcysteine, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 prevented the HNE-induced increased expression of cPLA(2) and p-cPLA(2). Immunocytochemical analysis revealed that staining for p-cPLA(2) in Ra2 cells was localized in the cytoplasm and more intense in the HNE-stimulated group than in the vehicle group. The present results provide in vitro evidence that HNE upregulates and phosphorylates cPLA(2) in microglia via the ERK and p38 MAPK pathways.

Alteration of angiogenic patterns on B16BL6 melanoma development promoted in Matrigel.

Because the progression and metastasis of solid tumors depend on their local microcirculation, we sought to characterize tumor angiogenesis three dimensionally in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into the subcutis in the skin on the back of syngeneic C57BL/6 mice. We found that B16 with Matrigel grew significantly faster than B16 alone and had altered tumor angiogenesis. Tumor vessels apparently grew vigorously in the opposite direction of the tumor without invading the tumor mass until at least day 10 of injection. In addition, vascular branching resulted not only from sprouting as was seen in B16 without Matrigel but also from vascular splitting, either because of compression from outside the vessels or from septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other tumor cells, including Lewis lung carcinoma and ASH-1 hybridoma cell lines, both of which were tested under the same conditions. The reduction in various angiogenic factors in Matrigel did not affect the angiogenic patterns and tumor growth. We hypothesize that tumor vessels may vigorously alter their angiogenic patterns in response to the local microenvironment.

Recruited peripheral blood monocytes participate in the liver extramedullary hematopoietic milieu.

The hematopoietic microenvironment has been investigated and well defined in the bone marrow. However, there is a lack of studies on the extramedullary hematopoietic milieu such as in the liver, to which hematopoietic stem cells migrate and there commence hematopoiesis under pathological conditions such as bone marrow failure. We induced extramedullary hematopoiesis by phenylhydrazine in the adult mouse liver and investigated the immunohistochemical, ultrastructural, and molecular changes within this organ. Using an intravital lectin injection technique, we found numerous monocytes attached to the central vein prior to hematopoietic foci formation. These cells were later incorporated into the hematopoietic foci. An increase in the mRNA expressions of the monocyte attracting chemokine CCL-2 (MCP-1) was noted in the central vein region as well as in cells within the hematopoietic foci. Together with local liver components, we regard these monocytes as components of the extramedullary hematopoietic milieu. We conclude that the recruitment of extra-hepatic monocytes is an important event during extramedullary hematopoiesis in the liver and that these monocytes participate in the liver hematopoietic microenvironment.