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.

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.

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.

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.

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.

Characterization of sinusoidal endothelial cells of the liver and bone marrow using an intravital lectin injection method.

The vascular endothelia express a variety of structural and biological phenotypes. We used an intravital injection method of plant derived lectins (Lycopersicon esculentum lectin (LEL), Ricinus communis Agglutinin-I (RCA-I), Ulex europaeus Agglutinin-I (UEA-I) and Concanavalin A (ConA)) to elucidate the morphology and function of the sinusoidal endothelium of the liver and bone marrow. All four lectins stained the sinusoidal endothelia of the liver and bone marrow in a patchy granular pattern which differed from the uniform and smooth staining pattern of non-sinusoidal vessels in other organs. By transmission electron microscopy, the granular pattern was identified as internalization of these lectins and subsequent accumulation within the endothelial cells, suggesting their active endocytosis. The endocytosis of these lectins emphasizes the fact that sinusoidal endothelial cells of the liver and bone marrow belong to the reticuloendothelial system (RES), a cell system characterized by internalization of foreign material. We introduce this intravital lectin injection as a useful technique to discriminate sinusoidal endothelial of the liver and bone marrow from other vascular endothelia.

Production of two novel monoclonal antibodies that distinguish mouse lymphatic and blood vascular endothelial cells.

We produced two novel rat monoclonal antibodies (LA102 and LA5) to identify mouse lymphatic vessels and blood vessels, respectively. We characterized the two antibodies as to the morphological and functional specificities of endothelial cells of both types of vessels. The antibodies were produced by a rapid differential immunization of DA rats with collagenase- and neuraminidase-treated mouse lymphangioma tissues. LA102 specifically reacted with mouse lymphatic vessels except the thoracic duct and the marginal sinus of lymph nodes, but not with any blood vessels. In contrast, LA5 reacted with most mouse blood vessels with a few exceptions, but not with lymphatics. LA102 recognized a protein of 25-27 kDa, whereas LA5 recognized a molecule of 12-13 kDa. Neither antibody recognized any currently identified lymphatic or vascular endothelial cell antigens. Immunoelectron microscopy revealed that the antigens recognized by LA102 and LA5 were localized on both luminal and abluminal endothelial cell membranes of each vessel type. Interestingly, LA102 immunoreactivity was strongly expressed on pinocytic or transport vesicle membrane in the cytoplasm of lymphatic endothelium. Besides endothelial cells, both antibodies also recognized some types of lymphoid cells. Since, the LA102 antigen molecule is expressed on some lymphoid cells, it may play important roles in the migration of lymphoid cells and in some transport mechanisms through lymphatic endothelial cells.

Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos.

The hemangioblast in the mesoderm gives rise to both angioblasts and hematopoietic stem cells. The movement of hemangioblast precursor cells in the fetal trunk is a critical event in early embryogenesis. Vascular endothelial growth factor (VEGF) signaling is likely involved in this migration given the partial disturbance of VEGF receptor (VEGFR)-positive cell accumulation and migration in VEGFR2 null mice or mice with a truncated VEGFR1. However, it is not clear how the VEGF system regulates this migration or its direction. We show here that the expression of VEGF-A is dominant in the anterior portion of the embryo, whereas VEGFR1 and VEGFR2 are expressed in the posterior portion of the embryo. An inhibitor of VEGFR kinase blocked the migration of VEGFR-positive cells in a whole-embryo culture system. In addition, VEGFR-positive cells migrated toward a VEGFR1- or VEGFR2-specific ligand in vitro. Furthermore, VEGFR-positive cells derived from wild-type or VEGFR2(+/-) mice moved rapidly anteriorly, whereas cells derived from VEGFR2(+/-) mice carrying a truncated VEGFR1 [VEGFR1(TM-TK)(-/-)] migrated little when injected into wild-type mice. These results suggest that the VEGF-A protein concentrated in the anterior region plays an important role in the guidance of VEGFR-positive cells from the posterior portion to the head region by interacting with VEGFR in the mouse embryo.

Evidence for recruitment of plasmacytoid dendritic cell precursors to inflamed lymph nodes through high endothelial venules.

Recruitment of dendritic cells (DCs) to lymph nodes (LNs) is pivotal to the establishment of immune response. Whereas DCs have been proven to undergo afferent lymphatic pathway to enter LNs from peripheral tissues, a question remains if DCs also migrate into LNs directly from the circulation. Here we demonstrate that plasmacytoid DC (pDC) precursors can transmigrate across high endothelial venules (HEVs) of inflamed LNs in mice. Bacterial infection induces a significant number of pDC and myeloid DC (mDC) precursors into the circulation. Both subsets express a common set of chemokine receptors except CXCR3, display parallel mobilization into the blood, but show distinct trafficking pathway to the LNs. In a short-term homing assay, whereas mDC precursors migrate to peripheral tissues and subsequently to draining LNs, pDC precursors directly enter the LNs in a CXCL9 and E-selectin dependent manner. Tumor necrosis factor-alpha controls not only DC precursor mobilization into the blood but also chemokine up-regulation on LN HEVs. A similar trafficking pathway is observed also in viral infection, and CXCR3(-/-) mice-derived pDC precursors show defective trans-HEV migration. This study clarifies the inflammation-dependent, chemokine-driven distinct property of DC precursor trafficking.

Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors.

Often described as incomplete or absent, the basement membrane of blood vessels in tumors has attracted renewed attention as a source of angiogenic and anti-angiogenic molecules, site of growth factor binding, participant in angiogenesis, and potential target in cancer therapy. This study evaluated the composition, extent, and structural integrity of the basement membrane on blood vessels in three mouse tumor models: spontaneous RIP-Tag2 pancreatic islet tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas. Tumor vessels were identified by immunohistochemical staining for the endothelial cell markers CD31, endoglin (CD105), vascular endothelial growth factor receptor-2, and integrin alpha5 (CD49e). Confocal microscopic studies revealed that basement membrane identified by type IV collagen immunoreactivity covered >99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1- micro m confocal optical sections, were <2.5 micro m in diameter and involved only 0.03% of the vessel surface. Despite the extensive vessel coverage, the basement membrane had conspicuous structural abnormalities, including a loose association with endothelial cells and pericytes, broad extensions away from the vessel wall, and multiple layers visible by electron microscopy. Type IV collagen-immunoreactive sleeves were also present on endothelial sprouts, supporting the idea that basement membrane is present where sprouts grow and regress. These findings indicate that basement membrane covers most tumor vessels but has profound structural abnormalities, consistent with the dynamic nature of endothelial cells and pericytes in tumors.

Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.

Endothelial cells of tumor vessels have well-documented alterations, but it is less clear whether pericytes on these vessels are abnormal or even absent. Here we report that alpha-smooth muscle actin (alpha-SMA) and desmin-immunoreactive pericytes were present on >97% of blood vessels viewed by confocal microscopy in 100-microm-thick sections of three different spontaneous or implanted tumors in mice. However, the cells had multiple abnormalities. Unlike pericytes on capillaries in normal pancreatic islets, which had desmin but not alpha-SMA immunoreactivity, pericytes on capillary-size vessels in insulinomas in RIP-Tag2 transgenic mice expressed both desmin and alpha-SMA. Furthermore, pericytes in RIP-Tag2 tumors, as well as those in MCa-IV breast carcinomas and Lewis lung carcinomas, had an abnormally loose association with endothelial cells and extended cytoplasmic processes deep into the tumor tissue. alpha-SMA-positive pericytes also covered 73% of endothelial sprouts in RIP-Tag2 tumors and 92% of sprouts in the other tumors. Indeed, pericyte sleeves were significantly longer than the CD31-immunoreactive endothelial cell sprouts themselves in all three types of tumors. All three tumors also contained alpha-SMA-positive myofibroblasts that resembled pericytes but were not associated with blood vessels. We conclude that pericytes are present on most tumor vessels but have multiple abnormalities, including altered expression of marker proteins. In contrast to some previous studies, the almost ubiquitous presence of pericytes on tumor vessels found in the present study may be attributed to our use of both desmin and alpha-SMA as markers and 100-microm-thick tissue sections. The association of pericytes with endothelial sprouts raises the possibility of an involvement in sprout growth or retraction in tumors.