Rat Endothelial Cell Antigen-1 Research Articles

Systemic Administration of Fluoro-Gold for the Histological Assessment of Vascular Structure, Integrity and Damage

Fluoro-Gold (F-G) has been used extensively as a fluorescent retrograde neuronal-track tracer in the past. We now report that intraperitoneal administration of 10 to 30 mg/ kg of F-G from 30 min to 7 days prior to sacrifice labels vascular endothelial cells of the brain, choroid plexus and meninges and can be used to assess vascular integrity and damage. F-G vascular labeling co-localized with rat endothelial cell antigen (RECA-1) in the membrane. F-G also intensely labeled the nuclei of the endothelial cells, and co-localized with propidium iodide staining of these nuclei. As well, the administration of F-G during neurotoxic insults produced by amphetamine, kainic acid or "penetrating" wound to the brain can detect where vascular leakage/ hemorrhage has occurred. Histological methods to detect F-G labeled brain vasculature were performed in the same manner as that used for fluorescent visualization of neuronal elements labeled with F-G after perfusion fixation and coronal sectioning (15 to 40 µm) of the brain. This in vivo F-G labeling of endothelial cells and their nuclei yields a clear picture of the integrity of the vasculature and can be used to detect changes in structure. Vascular leaks after "penetrating" wounds through the cortex and striatum, hyperthermic amphetamine exposure or excitotoxic kainate exposure were detected by F-G in the extracellular space and via parenchymal F-G subsequently labeling the terminals and neurons adjacent to the lesioned or damaged vasculature. Further studies are necessary to determine the extent of the leakage necessary to detect vasculature damage. Visualization of the F-G labeling of vasculature structure and leakage is compatible with standard fluorescent immuno-labeling methods used to detect the presence and distribution of a protein in histological sections. This method should be directly applicable to studying brain vascular damage that occurs in the progression of Alzheimer's disease, diabetes and for monitoring the brain vascular changes during development.

Stem cells from human exfoliated deciduous teeth enhance recovery from focal cerebral ischemia in rats

PurposeThe aim of this study was to investigate the effect of stem cells from human exfoliated deciduous teeth (SHED) after permanent MCAO (pMCAO). Materials and methodsAdult male Sprague-Dawley rats were subjected to pMCAO. After pMCAO, SHED was transplanted into the brain. Motor function and infarct volume were evaluated. Neurogenesis and vasculogenesis were determined using immunochemical markers. ResultsThe SHED group had more positive signals for doublecortin, neurofilament, anti-neuronal nuclei (NeuN) and rat endothelial cell antigen-1 (RECA1) in the peri-infarct area than the PBS group. Migration of doublecortin-positive neural progenitor cells (NPCs) from subventricular zone (SVZ) to the peri-infarct area was observed on day 16. Transplanted SHED merged vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) positive cells. ConclusionSHED promoted migration and differentiation of the endogenous NPCs and induced vasculogenesis, and ameliorated ischemic brain injury after pMCAO in rats.

Stem Cells from Human Exfoliated Deciduous Tooth-Derived Conditioned Medium Enhance Recovery of Focal Cerebral Ischemia in Rats

Regenerative therapy using stem cells is a promising approach for the treatment of stroke. Recently, we reported that dental pulp stem cells (DPSC) ameliorated ischemic tissue injury in the rat brain and accelerated functional recovery after middle cerebral artery occlusion (MCAO). In this study, we investigated the effects of stem cells from human exfoliated deciduous tooth (SHED)-derived conditioned medium (SHED-CM) on permanent MCAO (pMCAO). Adult male Sprague-Dawley rats were subjected to pMCAO. SHED-CM were then administered intranasally, and the motor function and infarct volume were evaluated. Neurogenesis and vasculogenesis were determined using immunochemical markers. The SHED-CM group had more positive signals than the Dulbecco's modified Eagle's medium group, with doublecortin (DCX), neurofilament H, neuronal nuclei, and rat endothelial cell antigen observed in the peri-infarct area. Migration of neuronal progenitor cells (NPC) with DCX from the subventricular zone to the peri-infarct area was observed on days 6 and 16, with migration on day 6 being the most prominent. In conclusion, SHED-CM promoted the migration and differentiation of endogenous NPC, induced vasculogenesis, and ameliorated ischemic brain injury after pMCAO as well as transplantation of DPSC.

MRI assessment of the intra‐carotid route for macrophage delivery after transient cerebral ischemia

The broad aim underlying the present research was to investigate the distribution and homing of bone marrow-derived macrophages in a rodent model of transient middle cerebral artery occlusion using MRI and ultrasmall superparamagnetic iron oxide (USPIO) to magnetically label bone marrow-derived macrophages. The specific aim was to assess the intra-carotid infusion route for bone marrow-derived macrophage delivery at reperfusion. Fifteen Sprague-Dawley rats sustained 1 h of middle cerebral artery occlusion. USPIO-labeled bone marrow-derived macrophages were slowly injected for 5 min immediately after reperfusion in ischemic animals (n=7), 1 h after the end of surgery in sham animals (n=5) and very shortly after anesthesia in healthy animals (n=3). Multiparametric MRI was performed at day 0, just after cell administration, and repeated at day 1. Immunohistological analysis included Prussian blue for iron detection and rat endothelial cell antigen-1 for endothelium visualization. Intra-carotid cell delivery brought a large number of cells to the ipsilateral hemisphere of the brain, as seen on both MRI and immunohistology. However, it was associated with high mortality (50%). The study of sham animals demonstrated that intra-carotid cell delivery could induce ischemic lesions and may thus favor additional brain damage. The present study highlights severe drawbacks to the intra-carotid delivery of macrophages at the time of reperfusion in this rodent model of transient cerebral ischemia. Multiparametric MRI appears to be a method of choice to monitor longitudinally the effects of cell infusion, allowing the assessment of both cell fate with the help of magnetic labeling and of potential tissue damage.

Annexin A2 acts as scaffolding receptor for plasminogen and tissue plasminogen activator in renal epithelia

Annexin A2 has been shown to act as a receptor for plasminogen (Plg) and its tissue type activator (tPA) in endothelial cells (EC). Aim of the present study was to elucidate the renal distribution of the components of this complex under control conditions and in a Thy1 nephritis model of proteinuric renal disease. Thy1 nephritis was induced by intravenous injection of OX7 anti‐Thy1 antibody (1mg/kg body weight). Distribution of Plg, tPA and ANXA2 was studied by immunofluorescence in controls and after 5 or 15 days of OX7 injection. Immunoreactive structures were further characterized by double‐labeling immunofluorescence using antibodies against rat endothelial cell antigen for EC, Na(+),K(+),2Cl(−) cotransporter for thick ascending limbs (TAL), aquaporin‐1 for thin descending limbs (tDL) and aquaporin‐2 for collecting ducts (CD). As expected, all three proteins were identified in EC of controls and Thy1 animals. In addition we found overlapping signal for tPA, Plg and ANXA2 in the basolateral and apical membrane of tDL, TAL and CD both, in treated and untreated animals. Furthermore, abundance of tPA and Plg in the apical membrane of TAL and CD was increased in proteinuric animals. In conclusion, our results provide evidence for the presence of a proteolytic complex in the apical membrane of renal epithelia where it may play a protective role in the setting of proteinuric disease.support: DFG FOR 667

Perfusion MRI for Monitoring the Effect of Sorafenib on Experimental Prostate Carcinoma: A Validation Study

The purpose of this study was to investigate with immunohistochemical validation whether dynamic contrast-enhanced MRI with small-molecule contrast medium is useful for monitoring the effects of the multikinase inhibitor sorafenib on prostate carcinomas in rats. Copenhagen rats (n = 20) into which prostate carcinoma (MAT-Ly-Lu-B2) had been implanted subcutaneously were imaged on the day of implantation and 7 days later with 3-T dynamic gadobutrol-enhanced MRI. The therapy group (n = 10) received daily administration of 10 mg/kg body weight sorafenib. Quantitative measurements of tumor perfusion, tumor vascularity, and permeability-surface area product were calculated with a two-compartment model. Dynamic contrast-enhanced MRI values were correlated with immunohistochemical results for validation. Tumor perfusion in sorafenib-treated prostate carcinoma declined significantly from day 0 to day 7 (47.9 ± 36.8 mL/100 mL/min to 24.4 ± 18.6 mL/100 mL/min; p < 0.05). No significant effect on permeability-surface area product was observed in either the therapy or the control group (p > 0.05). Tumor vascularity decreased significantly (p < 0.05) from day 0 to day 7 under sorafenib treatment (15.6% ± 11.4% to 5.4% ± 2.1%). Immunohistochemical analysis revealed significantly lower tumor vascularity in the therapy than in the control group (rat endothelial cell antigen 1, 74.4 ± 16.9 cells vs 197 ± 75.4 cells; p < 0.05). In sorafenib-treated tumors, significantly more apoptotic cells (terminal deoxynucleotidyl transferase-mediated nick end labeling, 6923 ± 3761 vs 3167 ± 1500; p < 0.05) and significantly fewer proliferating cells (Ki-67, 10,198 ± 3064 vs 15,003 ± 3674; p < 0.05) were observed than in the control group. Modest but significant correlations were observed between tumor perfusion and immunohistochemical tumor cell apoptosis (r = -0.56; p < 0.05) and between tumor perfusion and immunohistochemical tumor vascularity (r = 0.56; p < 0.05). Tumor perfusion quantified with gadobutrol-enhanced dynamic contrast-enhanced MRI can be used as a noninvasive surrogate parameter for monitoring the antiangiogenic, antiproliferative, and proapoptotic effects of sorafenib on prostate carcinoma allografts as validated with immunohistochemical analysis.

Age-related decline in label-retaining tubular cells: implication for reduced regenerative capacity after injury in the aging kidney

Recovery after acute kidney injury is impaired in the elderly, but the precise mechanism for such age-related incompetence remains unclear. By in vivo bromodeoxyuridine (BrdU) labeling, renal progenitor cells (label-retaining cells; LRCs) were identified in tubules of normal rat kidney and were shown to be the origin of proliferating cells after injury. In the present study, the involvement of LRCs in the age-related decline of tubular recovery after injury was examined. After 1 wk of BrdU labeling followed by a 2-wk chase period, ischemia-reperfusion injury was induced in 7-wk-, 7-mo-, and 12-mo-old rats. Age-related decreases in DNA synthesis and cell proliferation in renal tubules after injury were found. The number of LRCs also significantly declined with age. At 24 h after reperfusion, the number of LRCs significantly increased in all ages of rats tested. There was no significant difference in the ratio of LRC division among rats of different ages. The area of the rat endothelial cell antigen (RECA)-1-positive capillary network declined with age. When renal tubules isolated from rats treated with BrdU label were cocultured with human umbilical vein endothelial cells (HUVEC), the number of LRCs significantly increased compared with tubules cultured without HUVEC. These data suggest that the reduced capacity of tubular regeneration in the aging kidney is partly explained by the shortage of LRC reserves. The size of the LRC pool might be regulated by the surrounding peritubular capillary network.

Cavernous smooth muscle hyperplasia in a rat model of hyperlipidaemia‐associated erectile dysfunction

What's known on the subject? and what does the study add? Increased cavernous smooth muscle content has been repeatedly observed in rat models of hyperlipidaemia - associated erectile dysfunction. This study shows that the increased smooth muscle content is due to hyperplasia. • To investigate the structural changes, including possible smooth muscle hyperplasia, in the penis of a hyperlipidaemia-associated erectile dysfunction (ED) animal model. • Hyperlipidaemia was induced in rats through a high-fat diet. • Penile tissues of normal and hyperlipidaemic rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen, neuronal nitric oxide synthase (nNOS), and collagen type IV (Col-IV) before image and statistical analyses were carried out. • The main outcome measures were the smooth muscle, endothelial, Col-IV and nNOS content of the corpus cavernosum. • Phalloidin intensely stained all smooth muscle in the penis, revealing the circular and longitudinal components of cavernous smooth muscle (CSM). • The CSM content was significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). • Cell numbers in both circular and longitudinal CSM were significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). • Cavernous endothelial content was significantly lower in hyperlipidaemic than in normal rats (P < 0.05). • nNOS-positive nerves within the dorsal nerves, around the dorsal arteries, and in the corpora cavernosa were all significantly lower in the hyperlipidaemic than in the normal rats (P < 0.05). • Hyperlipidaemia is associated with reduced nNOS-positive nerves, reduced endothelium, and increased CSM in the penis. • The increased CSM is attributable to hyperplasia. • These structural changes may explain why hyperlipidaemic men are more likely to develop ED.

Improved Penile Histology by Phalloidin Stain: Circular and Longitudinal Cavernous Smooth Muscles, Dual-endothelium Arteries, and Erectile Dysfunction-associated Changes

To investigate whether fluorochrome-conjugated phalloidin can delineate cavernous smooth muscle (CSM) cells and whether it can be combined with immunofluorescence (IF) staining to quantify erectile dysfunction (ED)-associated changes. ED was induced by cavernous nerve crush in rats. Penile tissues of control and ED rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen (RECA), CD31, neuronal nitric oxide synthase (nNOS), and collagen-IV (Col-IV). Phalloidin was able to delineate CSM as composed of a circular and a longitudinal compartment. When combined with IF stain for CD31 or RECA, it helped the identification of the helicine arteries as covered by endothelial cells on both sides of the smooth muscle layer. When combined with IF stain for nNOS, it helped the identification that nNOS-positive nerves were primarily localized within the dorsal nerves and in the adventitia of dorsal arteries. When combined with IF stain for Col-IV, it helped identify that Col-IV was localized around smooth muscles and beneath the endothelium. Phalloidin also facilitated the quantitative analysis of ED-related changes in the penis. In rats with cavernous nerve injury, RECA or Col-IV expression did not change significantly, but CSM and nNOS nerve contents decreased significantly. Phalloidin stain improved penile histology, enabling the visualization of the circular and longitudinal compartments in the CSM. It also worked synergistically with IF stain, permitting the visualization of the dual endothelial covering in helicine arteries, and facilitating the quantification of ED-related histologic changes.

The effect of adipose-derived stem cells on augmentation ileocystoplasty: A pilot study

ObjectivesIncorporation of intestinal tissue into urinary tract elicits many metabolic and mechanical complications due to anatomical and physiological differences. Adipose-derived stem cells (ADSCs) improve vascularity and functional outcomes by a paracrine mechanism. In a pilot study we investigated whether ADSCs can survive in the augmented bladder and improve its function. Materials and methodsAutologous ADSCs were harvested from rat paragonadal fat and cultured before injection into a rat model of augmentation ileocystoplasty (study group). Control augmented bladders were injected with cell-free saline. Eight weeks later, rats underwent abdominal ultrasonography for upper tract changes and were examined by conscious cystometry to determine bladder function. After extirpation, augmented bladders were examined using Masson trichrome staining for connective tissue and muscle content, immunohistochemistry for α-smooth muscle actin, and rat endothelial cell antigen staining for endothelial cells. Changes in the extracellular matrix were assessed by determining the elastin content. ADSCs were labelled and tracked by 5-ethynyl-2-deoxyuridine nuclear staining. ResultsAbdominal ultrasonography showed better preservation of upper tract function in the ADSC group than in the saline-treated group (P=0.007). After 2months there were no differences in the variables assessed by conscious cystometry between the ADSC and saline-treated groups. However, the bladder weight was significantly greater in the ADSC-treated group. On immunohistochemistry, the implanted ADSCs survived up to 8weeks but did not transdifferentiate into smooth muscle or endothelial cells. ConclusionThese results suggested a potential role of ADSCs in modifying the intestinal segment in augmented bladders; this role has to be further elucidated.

Functional, Metabolic, and Morphologic Characteristics of a Novel Rat Model of Type 2 Diabetes-associated Erectile Dysfunction

To conduct a pilot study to investigate functional, metabolic, and penile morphologic changes in a novel model of lean DM2. Erectile dysfunction (ED) is a frequent sequela in patients with type 2 diabetes mellitus (DM2). Eight rats received a high-fat diet and 2 weeks later, 2 intraperitoneal injections of streptozotocin (STZ, 30 mg/kg). Five age-matched rats served as controls. Insulin challenge tests were performed at 6 and 12 weeks after induction of DM2. At 12 weeks, erectile function was tested by measurement of intracavernous pressure (ICP) increase upon cavernous nerve stimulation. Penile tissue and serum samples were harvested for histology and biochemistry, respectively. A lean DM2 model was established as demonstrated by decreased insulin resistance, elevated nonfasting plasma glucose levels, hyperlipidemia, and decreased insulin concentration in the absence of obesity. ICP/mean arterial pressure was significantly decreased in DM2 animals (0.29) compared with controls (0.81). Expression of neuronal nitric oxide synthase and rat endothelial cell antigen-1, and the smooth muscle/collagen ratio were significantly decreased in the penis of DM2 animals. We propose an inexpensive nongenetic animal model of lean DM2-associated ED. Microanatomical changes in the erectile tissue that reflect an advanced stage of the disease were observed.

Expression of the anti‐inflammatory protein Annexin A1 in the renal interstitium during the time course of acute Thy1 nephritis

Aim of the present study was to identify Annexin A1 (ANXA1) expressing renal interstitial cells under control conditions and during the course of acute Thy1 nephritis. ANXA1 immunoreactive interstitial cells were characterized by double labelling immunofluorescence using antibodies against ANXA1 and rat endothelial cell antigen for endothelial cells, 5′ecto-nucleotidase for interstitial fibroblasts, Major Histocompatibility Complex II for dendritic cells and CD68 for macrophages. In addition, ANXA1 expression of CD68 positive macrophages was studied during the time course of acute Thy1 nephritis (control, 24h, 5d, 15d). In control rats ANXA1 was detected in all interstitial fibroblasts, and in a subset of macrophages and endothelial cells. Dendritic cells were negative. Thy1 nephritis caused a transient glomerular infiltration of CD68 positive/ANXA1 negative macrophages with peak levels at 24h (610 +/− 126% of controls) and lower levels at the later time points (5d: 395 +/− 26% and 15d: 282 +/− 29% of control; p < .05 for each time point). The number of ANXA1 immunoreactive interstitial macrophages increased to 140 +/− 6% and to 175 +/− 21% of controls on d5 and d15 (p < .05). We have thus shown that ANXA1 is produced by numerous renal interstitial cell types of healthy rats and that infiltrating macrophages may be an important source of renal ANXA1 during the resolution phase of renal inflammatory disease. Work was supported by the German Research Foundation (FOR 667).

Effect of Vasopressin on the renal distribution of Annexin A2 in Brattleboro rats

Aim of the present study was to elucidate the renal expression pattern of Annexin A2 (ANXA2) during steady state and during activation of the urine concentration mechanism. To this end, adult Brattleboro rats (DI rats) were treated for 3d with the selective V2 vasopressin receptor agonist desmopressin (dDAVP, 5ng/h) or vehicle via osmotic minipump. Renal ANXA2 distribution was studied by double-labelling immunofluorescence using two different antibodies against ANXA2 and antibodies against rat endothelial cell antigen for endothelial cells, Tamm-Horsfall protein for thick ascending limbs (TAL) and aquaporin-2 for collecting ducts (CD). In untreated DI rats strong ANXA2 immunoreactivity was detected in cells of the Bowman capsule and in endothelial cells. Cells of the CD showed intermediate staining throughout the cytosol. In addition we observed a weak but consistent ANXA2 signal in the apical membrane of TAL profiles. Treatment of DI rats with dDAVP caused a marked shift of ANXA2 protein from the cytosol of CD cells to the apical and the basolateral membrane. In summary, we have shown that ANXA2 is expressed in cells of the Bowman capsule, the renal endothelium, the TAL and in the collecting duct where it may be involved in vasopressin signalling. This study was supported by the German Research Foundation (FOR 667).

A novel quantification of blood–brain barrier damage and histochemical typing after embolic stroke in rats

Treatment strategies in acute ischemic stroke are still limited. Considering numerous translation failures, research is tending to a preferred use of human-like animal models, and a more-complex perspective of tissue salvaging involving endothelial, glial and neuronal components according to the neurovascular unit (NVU) concept. During ischemia, blood–brain barrier (BBB) alterations lead to brain edema and hemorrhagic transformation affecting NVU components. The present study aims on a novel quantification method of BBB damage and affected tissue following experimental cerebral ischemia, closely to the human condition. Wistar rats underwent embolic middle cerebral artery occlusion, followed by an intravenous application of fluorescein isothiocyanate (FITC)-tagged albumin (≈ 70 kDa) and/or biotinylated rat IgG (≈ 150 kDa) as BBB permeability markers. Both fluorescent agents revealed similar leakage and allow quantification of BBB permeability by fluorescence microscopy, and after immunohistochemical conversion into a permanent diaminobenzidine label at light-microscopical level. The following markers were identified for sufficient detection of NVU components: Rat endothelial cell antigen-1 (RECA) and laminin for vessels, Lycopersicon esculentum and Griffonia simplicifolia agglutinin for vessels and microglial subpopulations, ionized calcium binding adaptor molecule 1 (Iba1), CD68 and CD11b for macrophages, activated microglia, monocytes and neutrophils, S100β for astroglia, as well as NeuN and HuC/D for neurons. This is the first report confirming the usefulness of simultaneously applied FITC-albumin and biotinylated rat IgG as BBB permeability markers in experimental stroke, and, specifying antibodies and lectins for multiple fluorescence labeling of NVU components. Newly elaborated protocols might facilitate a more-complex outcome measurement in drug development for cerebral ischemia.

The effects of low-frequency pulsed electromagnetic fields on microcirculation angiogenesis in acute hindlimb ischemia among diabetic rats

Objective To observe the effects of low-frequency pulsed electromagnetic fields (LFPEMFs) on microcirculation angiogenesis in the hindlimbs of diabetic rats with acute ischemia. Methods Models of acute hindlimb ischemia were established in 60 male Sprague-Dawley diabetic rats. The diabetes model was established using 60 mg/kg intraperitoneal injections of streptozotocin (STZ). Fasting blood glucose levels were greater than 300 mg/dL. The rats were randomly divided into experimental and control groups. The rats in the experimental group were exposed to low-frequency pulsed electromagnetic fields for 2 hours each day, while the control group was not given any treatment. Laser-Doppler perfusion was used to measure blood flow in the ischemic hindlimb on days 0,7, 14 and 28 after the operation. The immunofluorescence of rat endothelial cell antigen-1 ( RECA-1) was used to evaluate the changes in angiogenesis. The levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) were determined by both Western blotting and ELISA, and VEGFR2 and FGFR1 levels in the ischemic skeletal muscle were determined by Western blotting on days 7, 14 and 28 after the operation. Results The average perfusion ratio was significantly greater in the experimental group at days 14 and 28 compared with the control group. RECA-1 density in the tissues had increased significantly in the experimental group at the 14th and 28th day. The same was observed for FGF-2 and its receptor, but there was no significant difference for VEGF or its receptor in either group. Conclusions LFPMEFs can promote angiogenesis in acute hindlimb ischemia of diabetic rats by up-regulating FGF-2. This suggests that LFPMEFs may be useful for preventing and treating lower limb ischemia in diabetic humans. Key words: Low-frequency pulsed electromagnetic fields; Ischemia; Angiogenesis; Endothelial cell antigen-1 ; Fibroblast growth factor-2

Effects of cyclohexenonic long-chain fatty alcohol in type 2 diabetic rat nephropathy

We attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol (CHLFA) on the alterations of type 2 diabetes-induced nephropathy. Forty-week-old male Goto-Kakizaki (GK) and Wistar rats were divided into four groups of 6 to 8 animals. Group A consisted of eight Wistar rats and served as an age-matched control group. Group B (7 GK rats) received no treatment and served as a diabetic group. Group C (6 GK rats) was treated daily with low-dose CHLFA (2 mg/ kg/body weight, subcutaneously) for 30 weeks, and Group D (6 GK rats) with high-dose CHLFA (8 mg/kg/body weight) for 30 weeks. At the end of the treatment period, urinary protein excretion, blood chemistry, renal histological, and immunohistological analyses were conducted. Although CHLFA administration did not influence serum glucose or insulin levels, it reversed diabetes-induced increases in urinary protein excretion and serum creatinine. Light microscopically, CHLFA treatment ameliorated the otherwise elevated glomerular sclerotic scores in the diabetic group.Immunohistochemically, increased expression of desmin and decreased expression of rat endothelial cell antigen-1 in the group with untreated diabetes both showed a reversal to control levels in the high-dose CHLFA treatment group. In conclusion, CHLFA may ameliorate type 2 diabetes-induced nephropathy.

Effects of cold preservation and reperfusion injury on the regeneration of donor liver and associated mechanisms.

Objective To investigate the effects of cold preservation and reperfusion injury on the regen-eration of donor liver and to study the mechanisms. Methods Male SD rats were divided in to sham group (6 rats), UW 1 h group (48 rats) and UW 12 h group (48 rats). Liver tissue specimens were collected at different time points after orthotopic liver transplantation or sham operation. The morphology of liver tissue was observed via light microscope and transmission electron microscope. Proliferation of hepatocytes and sinusoidal endothelial cells (SECs) were assessed by a double immunostaining technique using antibodies against rat endothelial cell antigen-1 and bromodeoxyuridine (BrdU). Expression of vascular endothelial growth factor (VEGF) and its receptors, fins-like tyrosine kinase-1 (flt-1) and fetal liver kinase-1 (flk-1) was evaluated by immunohistochemistry. The mRNA expression of flt-1 was detected by a RT-PCR method. Mean comparison in groups was conducted by one-way ANOVA or t test. Results BrdU labeling indexes of hepatocytes and SECs in UW 12 h group was significantly higher than those in UW 1 h group (F = 61.45,41.4, P < 0.05). The proliferation of hepatocytes peaked at 48 h after operation in both UW 1 h group and UW 12 h group. However, the proliferation of SECs was fallen behind compared to hepatocytes, with a peak appeared at 72 h in UW 1 h group and at 96 h in UW 12 h group, respec-tively. The expression of VEGF was up-regulated in both UW 1 h group and UW 12 h group compared to sham group. Furthermore, expression of flt-1 and flk-1 was found to be mainly limited in SECs, with a peak in expres-sion occurring between 72 h and 96 h, coinciding with the peak in SECs proliferation in UW 1 h group. Conversely, flt-1 was found to be reduced significantly on mRNA level at any time throughout the experiment in UW 12 h group compared to sham group (F = 141.67, P < 0.05). Conclusion Reduced expression of flt-1 results in a retarded regeneration of SECs, and then the recovery of rat donor liver function is delayed after cold-preserved transplantation. Key words: Liver transplantation; Sinusoidal endothelial cells; Liver regeneration

Vascular changes in epilepsy: functional consequences and association with network plasticity in pilocarpine-induced experimental epilepsy

Angiogenesis and blood-brain-barrier (BBB) damage have been proposed to contribute to epileptogenesis and/or ictogenesis in experimental and human epilepsy. We tested a hypothesis that after brain injury angiogenesis occurs in the most damaged hippocampal areas with the highest need of tissue repair, and associates with formation of epileptogenic neuronal networks. We induced status epilepticus (SE) with pilocarpine in adult rats, and investigated endothelial cell proliferation (BrdU and rat endothelial cell antigen-1 (RECA-1) double-labeling), vessel length (unbiased stereology), thrombocyte aggregation (thrombocyte immunostaining), neurodegeneration (Nissl staining), neurogenesis (doublecortin (DCX) immunohistochemistry), and mossy fiber sprouting (Timm staining) in the hippocampus at different time points post-SE. As functional measures we determined BBB leakage (quantified immunoglobulin G (IgG) immunostaining), and hippocampal blood volume (CBV) and flow (CBF) in vivo (magnetic resonance imaging, MRI). The total length of hippocampal blood vessels was decreased by 17% at 2 d after status epilepticus (SE) induced by pilocarpine in adult rats ( P<0.05 as compared to controls) which was not accompanied by alterations in hippocampal blood volume (BV) and flow (BF). Number of proliferating endothelial cells peaked at 4 d post-SE and correlated with an increase in vessel length ( r=0.900, P<0.05). Vessels length had recovered to control level or even higher at 2 wk post-SE, angiogenesis being most prominent in the CA3 (128% as compared to that in controls, P<0.05), and was associated with increased BV (178% as compared to that in controls, P<0.05). Enlargement of vessel diameter in the hippocampal fissure was associated with thrombocyte aggregation in distal capillaries. BBB was most leaky during the first 4 d post-SE and increased IgG extravasation was observed for 60 d. Our data show that magnitude of endothelial cell proliferation is not associated with severity of acute post-SE neurodegeneration or formation of abnormal neuronal network. This encourages identification of molecular targets that initiate and maintain specific aspects of tissue reorganization, including preservation and proliferation of endothelial cells to reduce the risk of epileptogenesis and enhance recovery after brain injury.

Detection and characterization of circulating endothelial progenitor cells in normal rat blood

Introduction There are currently few widely accepted noninvasive detection methods for drug-induced vascular damage. Circulating endothelial progenitor cell (EPC) enumeration in humans has recently gained attention as a potential biomarker of vascular injury/endothelial damage/dysfunction. The rat is commonly used in preclinical drug development toxicity testing and lacks consensus noninvasive methodologies for immunophenotypic identification of EPCs. Identification of immunophenotypic markers of EPCs in the rat would enable transfer of technologies use`d in human for potential development of biomarkers for vascular injury the rat. Therefore, the aim of this work was to develop methods to consistently identify a discreet population of EPCs from rat peripheral blood. Methods EPCs were identified phenotypically from rat blood using cell culture, immunolabeling, fluorescence microscopy, and flow cytometry. EPCs isolated using immunolabeling coupled with magnetic separation and flow cytometric cell sorting were characterized genotypically using mRNA analysis. Results A modified colony forming unit (CFU)-Hill assay confirmed existence of immature EPCs in peripheral blood. Extended in vitro culture resulted in a morphology and immunophenotype consistent with mature endothelial cells as noted by positive staining for CD31, von Willebrand factor, rat endothelial cell antigen, and negative staining for smooth muscle cell α-actin. The majority of the cells identified as LDL +/CD11b/c − did not stain positively for either vWF or CD31. EPC populations isolated using magnetic separation and cell sorting were consistently positive for PECAM1, EDN1, FLK1, VWF, ITGAD, CCR1, IP30, and MMP2 mRNA expression. Cells identified as EPCs express cell-surface and gene expression markers consistent with endothelial cells and endothelial progenitor cell populations. Discussion Vascular trauma induces transient mobilization of EPCs in humans and their enumeration and characterization have been proposed as a surrogate biomarker for assessment of vascular injury. Potential exists for using rat circulating EPCs as a surrogate sampling population for biomarker development in drug-related injury in preclinical toxicity studies. A prerequisite to biomarker development is the ability to consistently identify a discreet population of EPCs from peripheral rat blood. This work describes novel methods for isolation and validation of phenotypically and genotypically consistent populations of rat EPCs from peripheral blood. These methods are well suited for potential future use in validation of enumeration and/or biomarker development methods in the rat.

Fibroblast Growth Factor 2 Promotes Endothelial Differentiation of Adipose Tissue-Derived Stem Cells

Adipose tissue-derived stem cells (ADSC) could potentially restore endothelial function in vasculogenic erectile dysfunction (ED). The mechanism for ADSC endothelial differentiation remained unidentified. To test whether ADSC could differentiate into endothelial cells in the penis and to identify the underlying mechanism of ADSC endothelial differentiation. For in vivo endothelial differentiation, ADSC were labeled with bromodeoxyuridine (BrdU), injected into rat corpora cavernosa, and localized by immunofluorescence and phase-contrast microscopy. For in vitro endothelial differentiation, ADSC were grown in endothelial growth medium 2 (EGM2), stained for endothelial markers CD31, von Willebrand Factor (vWF), and endothelial nitric oxide synthase (eNOS), and assessed for the ability to form tube-like structures in Matrigel and to endocytose acetylated low-density lipoprotein (Ac-LDL). To identify factors that promote ADSC endothelial differentiation, ADSC were grown in various media, each of which contained a specific combination of supplemental factors and assessed for LDL-uptake. PD173074, a selective inhibitor of fibroblast growth factor 2 (FGF2) receptor, was used to confirm the importance of FGF2 signaling for ADSC endothelial differentiation. In vivo endothelial differentiation was assessed by immunofluorescence microscopy. In vitro endothelial differentiation was assessed by immunofluorescence, Matrigel tube formation, and Ac-LDL uptake. Injected ADSC were localized to the sinusoid endothelium, some of which stained positive for both BrdU and endothelial antigen rat endothelial cell antigen. ADSC proliferated at a faster rate in EGM2 than in standard DMEM, expressed endothelial markers CD31, vWF, and eNOS, formed tube-like structures in Matrigel, and endocytosed Ac-LDL. These properties were greatly diminished when ADSC were grown in the absence of FGF2 but were unaffected when grown in the absence of vascular endothelial growth factor, insulin-like growth factor, or epidermal growth factor. Furthermore, ADSC displayed similar endothelial properties when grown in FGF2-supplemented basic medium as in EGM2. Finally, blockade of FGF2 signaling with PD173074 abrogated ADSC endothelial differentiation. ADSC could differentiate into endothelial cells in the penis. FGF2 signaling mediates ADSC endothelial differentiation.