Total Area Of Blood Vessels Research Articles

Abstract 6592: An AI platform for vascular and fibrosis analysis of pathology images

Abstract Introduction: Changes in the number of blood vessels and the proportion of fibrosis regions in xenograft tumors are key indicators for efficacy of oncology drugs, which currently are assessed mainly by human visual examination that is time-consuming, inaccurate and biased because only part of a pathology image is checked. We developed an online AI platform that integrates machine learning algorithms in Computer Vision to quickly and automatically obtain accurate and comprehensive information on blood vessels and fibrosis regions from a whole pathology image. Methods: In a stained pathology image, blood vessels are stained with a different color than other components (e. g. connective tissues, tumor cells, background), so we can directly locate pixels representing them. They also have different topological structures, so we can outline and count the contours of vessels. To count the number of vessels more precisely and reduce the interference of noise, we keep a contour only if it intersects with some located pixels and the number of pixels in the intersection exceeds an adjustable threshold. The fibrosis regions are identified by color contrast since they are stained with a distinct and easily recognizable color. The algorithms were implemented using the OpenCV package. Results: Our AI platform automatically annotates blood vessels and fibrosis regions in pathology images, and generates reports in excel format to report the number and total area of blood vessels, the area and ratio of fibrosis regions, and execution time. It demonstrates superior performance to standard pathology software. Citation Format: Yawen Zheng, Likun Zhang, Dawei Wang, Sheng Gao. An AI platform for vascular and fibrosis analysis of pathology images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6592.

Interleukin-17 plays a role in dental pulp inflammation mediated by zoledronic acid: a mechanism unrelated to the Th17 immune response?

To evaluate the influence of RORγT inhibition by digoxin on inflammatory changes related to interleukin-17 (IL-17) in the pulp of rats treated with zoledronate (ZOL). Forty male Wistar rats were divided into a negative control group (NCG) treated with saline solution, a positive control group (PCG) treated with ZOL (0.20 mg/kg), and three groups treated with ZOL and co-treated with digoxin 1, 2, or 4 mg/kg (DG1, 2, and 4). After four intravenous administrations of ZOL or saline solution in a 70-day protocol, the right molars were evaluated by histomorphometry (number of blood vessels, blood vessels/µm2, cells/µm2, total blood vessel area, and average blood vessel area) and immunohistochemistry (IL-17, TNF-α, IL-6, and TGF-β). The Kruskal-Wallis/Dunn test was used for statistical analysis. PCG showed an increase in total blood vessel area (p=0.008) and average blood vessel area (p=0.014), and digoxin treatment reversed these changes. DG4 showed a reduction in blood vessels/µm2 (p<0.001). In PCG odontoblasts, there was an increase in IL-17 (p=0.002) and TNF-α (p=0.002) immunostaining, and in DG4, these changes were reversed. Odontoblasts in the digoxin-treated groups also showed an increase in IL-6 immunostaining (p<0.001) and a reduction in TGF-β immunostaining (p=0.002), and all ZOL-treated groups showed an increase in IL-17 (p=0.011) and TNF-α (p=0.017) in non-odontoblasts cells. ZOL induces TNF-α- and IL-17-dependent vasodilation and ectasia, and the classical Th17 response activation pathway does not seem to participate in this process.

Local Adenoviral Delivery of Vascular Endothelial Growth Factor C Induces Lymphangiogenesis in the Conjunctiva in Rabbits

Introduction: The purpose of this study was to determine if conjunctival lymphangiogenesis can be induced using adenoviral delivery of vascular endothelial growth factor C (VEGF-C). Methods: Seventeen New Zealand white rabbits received a subconjunctival injection containing 3.5 × 107 plaque-forming units of an adenoviral vector containing the gene-encoding VEGF-C (Ad-VEGF-C). The contralateral eye was used for control experiment (the same volume of either saline or an empty vector). After 2 weeks, the animals were examined with trypan blue conjunctival lymphangiography, and the eyes were harvested for histology and immunohistochemistry (podoplanin and CD31). Results: Trypan blue conjunctival lymphangiography revealed significantly more extensive conjunctival vessel network in the Ad-VEGF-C group compared with control: 1.35 ± 0.67 versus 0.28 ± 0.17 vessel length/analysed area (p = <0.0001). This finding was confirmed with immunohistochemistry, where a significant increase in the number of lymphatic vessels was found compared to control; 34 ± 9 per mm2 versus 13 ± 8 per mm2 (p = 0.0019). Furthermore, there was a significant increase in lymphatic cross-sectional area; 32,500 ± 7,900 µm2 per mm2 versus 17,600 ± 9,700 µm2 per mm2 (p = 0.0149). Quantification of blood vessels revealed no significant difference in blood vessel density between Ad-VEGF-C and control; 19 ± 9 per mm2 versus 14 ± 8 per mm2 (p = 0.1971). There was no significant difference in total blood vessel area; 13,200 ± 7,600 µm2 per mm2 versus 7,100 ± 3,000 µm2 per mm2 (p = 0.0715). Eyes treated with an adenoviral vector (VEGF-C or empty vector) responded with a reactive cellular response, predominantly lymphocytes, towards the vector. Conclusion: The study demonstrates the feasibility of inducing conjunctival lymphangiogenesis with a single subconjunctival injection of Ad-VEGF-C. Future studies will explore how this can be used with a therapeutic purpose.

Newly formated (tertiary) lymphoid structures in tumor growth

Aim to reproduce tertiary lymphoid structures and study their features in a malignant neoplasm in the experiment.&#x0D; Material and methods. Male white rats of the Wistar stress-resistant line were inoculated with the tumor strain sarcoma M1. Using the methods of microsurgical preparation, color lymphography and ultrasonic sonolipodestruction we isolated the lymph nodes without metastatic lesions (described as secondary in normal anatomy), the lymph nodes with metastases of M1 sarcoma, and the newly formed nodes (extrauterine or tertiary). Lymph nodes were examined macroscopically, micromorphometric studies were performed, histological and immunohistochemical signs of tertiary lymph nodes were determined. The obtained data were subjected to statistical analysis.&#x0D; Results. In 60% of rats, in the right axillary region, in addition to the secondary lymph nodes described in normal anatomy, the tertiary lymph nodes were found on the side of the tumor. They were located near the histologically verified metastatic lymph node. The studied parameters of the main structural and functional zones of the tertiary lymph nodes had the following features: the enlarged marginal sinuses; a fewer number of lymphoid follicles; the average total area of blood vessels increased by 92% in relation to the metastatic lymph nodes and by 78% in relation to the secondary lymph nodes. The significant differences were registered with CD3, CD4, CD20, CD30 markers.&#x0D; Conclusion. The study proved the possibility of reproducing tertiary lymphoid nodes in the experiment with malignant growth and revealed a number of their features.

The effect of spring diameter on porcine ileal distraction enterogenesis.

Spring-mediated distraction enterogenesis has proven to be successful for intestinal lengthening. We aimed to evaluate the effect of spring diameter mismatch on intestinal adaptation. Juvenile mini-Yucatan pigs underwent placement of compressed nitinol springs with diameter of 10, 11, or 12mm into the ileal lumen. Pigs were euthanized on postoperative day 7. The lengths, histology, total area of blood vessels, and enteric ganglia were evaluated. All spring groups exhibited significant ileal lengthening. Across the different diameters, spring-expanded segments were similar in terms of ileal lengthening, crypt height, muscular thickness, blood vessels, and enteric ganglia area. Spring-mediated distraction enterogenesis is successful in the porcine ileum. A smaller diameter spring is as effective as a larger diameter spring in lengthening the ileum. Springs of varying diameters result in comparable structural changes in the ileum.

QS16. The Effects of Variations in the Skin Surface Temperature on Capillary Blood Flow Using Video-capillaroscopy

Purpose: Video-capillaroscopy (VC) can be used to evaluate real-time flow of red blood cells in capillaries at a depth of 1-mm from the skin surface. Therefore, this device could be used in microsurgery to assess the perfusion of flaps, replanted fingers, skin grafts, and keloids. The details of capillary changes during skin cooling under VC observations are not known. By investigating the adaptations of capillaries after cooling using VC, we determined the effects of temperature on skin capillaries with those at room temperature, establishing temperature criteria for future VC applications. Therefore, we observed and compared VC findings on skin areas often used for flap harvest at a normal and lower body temperature. Methods: Twenty healthy Japanese adults were included. Skin capillaries were observed at the lateral thigh, forearm, mid-axillary line, abdomen, and fingertip using VC (GOKO Bscan-Z). Ice packs were used to lower the skin temperature to less than 35°C. The VC findings were recorded for three minutes before and after cooling. By means of the ImageJ software, we measured the total blood vessel area (pixels) as a percentage of the total area of the visual field (pixels). Also, we measured the blood flow velocity (μm/s) using GOKO-VIP software and the results for both temperatures (normal and after cooling) were then compared. Results: The average age and BMI were 37.5±9.72 years and 22.4±2.45 kg/m2 respectively. The mean arterial pressure was 89.4±3.1 mmHg. In accordance with the Fitzpatrick phenotype, eleven patients were type II, five were type III, and four were type IV. The average skin temperature before and after cooling were 36.4±0.2°C and 34.7±0.8°, respectively. The blood vessel areas were significantly different before and after cooling in all anatomical areas (p <.001). The average reduction rate of the vessel area after cooling was 63% for the thigh, 30% for the forearm, 43.3% for the mid-axillary line, 35% for the abdomen, and 65% for fingertips. The mean reduction rate of the vessel area was significantly different among anatomic regions (p <.001). The average blood flow velocities were significantly different before and after cooling in all anatomical areas (p <.001). The average blood flow velocity reduction rate after cooling was 75.7% for the thigh, 55.3% for the forearm, 68.9% for the mid-axillary line, 61.6% for the abdomen, and 79.2% for fingertips. The average blood flow velocity reduction rate after cooling was significantly different among anatomic regions (p <.001). Conclusion: Decrease in skin surface temperature resulted in capillary vasoconstriction and a decrease of capillary blood flow velocity in all areas. When VC is used for flap monitoring, it is important to keep the observation area warm since decreasing the temperature in the monitored area might result in the false diagnosis of arterial occlusion.

Relationship Between Carotid Artery Angle and Plaque Morphology in Acute Cerebral Infarction Patients.

Background:High-resolution magnetic resonance imaging (HRMRI) can be used to clearly distinguish the luminal structure of the carotid artery (CA); measure the lumen, vessel wall, and total blood vessel area; and accurately describe the morphologic features of plaques.Methods:This study used HRMRI to investigate the relationship between geometric features and volume and composition of carotid atherosclerotic plaques. Patients (n=81) who had experienced acute cerebral infarction (CI) within 7±3 days before admission to the Neurology Department of Beijing Tiantan Hospital between November 2011 and June 2012 were enrolled in the study. CA HRMRI was performed to analyze the geometry and morphology of plaques in 160 blood vessels.Results:The median left and right internal carotid artery angles (ICAAs) were 32.79 and 31.00 degrees, respectively. Aside from the nonplanar external CA angle, plaque volume and angles did not differ significantly between the left and right sides. Age (B=3.77; P=0.03) and nonplanar ICAA (B=4.70; P=0.01) were predictors of left but not right carotid plaque volume. ICAA and bifurcation angle did not predict plaque volume.Conclusions:In this study, CA morphology in acute stroke patients is not associated with plaque volume or composition, but age and nonplanar ICAA can predict left carotid plaque volume.

Effect of Fascia Implantation and Controlled Release of Basic Fibroblast Growth Factor for Muscle Atrophy in Rat Laryngeal Paralysis.

To improve lateral thyroarytenoid (TA) muscle atrophy after laryngeal paralysis, reconstruction of the vascular network of the atrophied muscle is necessary. We therefore evaluated whether the controlled release of basic fibroblast growth factor (bFGF) with autologous fascia implantation could affect vascular reconstruction in the lateral TA muscle. Animal experiment. Laboratory. Unilateral laryngeal paralysis was induced in 20 rats. The rats were implanted with autologous fascia and a gelatin hydrogel sheet with or without 1 µg of bFGF (fascia and bFGF + fascia groups; n = 5 each) and with only a gelatin hydrogel sheet with bFGF (bFGF group: n = 5). Another group remained untreated (n = 5) at 4 months after paralysis. At 3 months since transplantation, intra- and intergroup comparisons of the muscle volumes and total area of blood vessels in the lateral TA muscle were performed. When compared with the untreated group, the bFGF + fascia group showed a significant increase in muscle volume (P =.0008) and vascular area (P =.0002) in the lateral TA muscle, whereas the other 2 treated groups demonstrated an insufficient effect. bFGF + fascia implantation showed histologic improvement in severe laryngeal paralysis. We demonstrated that the decrease in lateral TA muscle mass after paralysis might be countered by the reconstruction of the vascular network. Our findings indicate that hypovascular and denervated areas of the laryngeal muscle can be regenerated by the implantation of growth factors and scaffolds with surgical stress. 5.

Risk assessment of first upper gastrointestinal bleeding using computerized tomoscanning in esophageal varices patients with cirrhosis and portal hypertension.

To evaluate the risk of first upper gastrointestinal bleeding by computerized tomoscanning (CT) for esophageal varices patients with cirrhotic portal hypertension.One hundred thirty two esophageal varices patients with cirrhotic portal hypertension who are also complicated with gastrointestinal bleeding were recruited as bleeding group, while another 132 patients without bleeding as non-bleeding group. The diameter of esophageal varices, number of vascular sections, and total area of blood vessels were measured by CT scanning. The sensitivity and specificity of these indicators were calculated, and Youden index was adjusted with the critical point.The diameter of esophageal varices was 7.83 ± 2.76 mm in bleeding group, and 6.57 ± 3.42 mm in non-bleeding group. The Youden index was 0.32 with the critical point 5.55 mm. The area under the receiver operating characteristics (AUROC) was 0.72. The number of venous vessels was 4.5 ± 2 in bleeding group, whereas being 4 ± 2 in non-bleeding group. The Youden index was 0.35 with a critical point 4, and the area under the curve (AUC) was 0.68. The blood vessel area was 1.73 ± 1.15 cm2 in bleeding group, and 1.12 ± 0.89 cm2 in non-bleeding group. The Youden index was 0.48 with the critical point being 1.03 cm2, and corresponding AUC was 0.82.Among all 3 indicators of the total area, diameter, and number of sections of the esophageal varices, the total area of esophageal varices showed more accuracy as a potential and novel indicator for bleeding prediction.

Carotid artery calcification score and its association with cognitive impairment.

PurposeTo retrospectively investigate the possible association between carotid artery calcification score (CS) and cognitive impairment in carotid artery stenosis (CAS) patients.Patients and methodsCarotid artery was measured in 102 patients with cervical carotid arteries using Color Doppler ultrasound, multi-detector row spiral CT angiography and MRI scanning. Correlation analysis between CSs obtained by MD CT and cognitive scores was performed, and the correlation between CSs and vascular stenosis degree and MRI-measured plaque histological (lipid-rich necrotic nucleus [LRNC], intraplaque hemorrhage and fibrous cap surface rupture) and morphological parameters (lumen area [LA], wall area [WA], total area of blood vessels [TVA], plaque burden [PB]) was analyzed. Follow-up review analysis was conducted on 38 postoperative patients.ResultsSignificant negative correlation was discovered between CS value and cognitive scores in CAS patients (R=−0.359, P<0.001), which did not exist in postoperative patients (P=0.348); CS value also showed significant correlation with WA (R=0.521, P=0.042), TVA (R=0.215, P=0.017) and PB (R=0.237, P=0.003) and had a certain predictive value for the occurrence probability of carotid plaque LRNC (P=0.029, AUC =0.780) in preoperative patients.ConclusionCarotid artery CSs have significant correlation with cognitive scores, which could be used as risk factor for early screening of cognitive impairment in CAS patients. The possible mechanism may be related to the calcification impact on the plaque burden.

Overexpression of MicroRNA-1 in Prostate Cancer Cells Modulates the Blood Vessel System of an In Vivo Hen's Egg Test-Chorioallantoic Membrane Model.

In prostate cancer (PC), the formation of new blood vessels is stimulated by hypoxic conditions, androgens, and a number of molecular factors including microRNAs. MicroRNA-1 (miR-1) has been characterized in some tumor entities as anti-angiogenic, but this has not yet been investigated in PC. PC cells stably overexpressing miR-1 (LNCaP-miR-1) were incubated on an in vivo hen's egg test-chorioallantoic membrane (HET-CAM) model and compared to maternal LNCaP cells. Cell growth, blood vessel organisation, and total blood vessel area were analysed. Both matrigel-embedded LNCaP and LNCaP-miR-1 cells formed compact tumor-like cell aggregates on the CAM of the HET-CAM model. Although not quantifiable, bleeding of the CAM and remodelling of the blood vessel network in the CAM indicated an influence of miR-1 on the vascular system. The statistically significant decrease in the total surface area of blood vessels in the visible CAM section to 79.4% of control cells demonstrated the antiangiogenic properties of miR-1 for the first time. MiR-1 had a tumor-suppressive and anti-angiogenic effect in an in vivo PC model. In the clinic, miR-1-mediated anti-angiogenesis would result in reduced tumor supply and increased hypoxic stress inside the tumor. Thus, miR-1 restoration by nucleic acid-based miR-1 mimetics would represent a promising option for future PC therapy.

Angiogenesis in CD5-positive Diffuse Large B Cell Lymphoma: A Morphometric Analysis.

CD5-positive diffuse large B cell lymphoma (DLBCL) is the least frequent immunohistochemical subgroup of DLBCL. The relatively little available data suggests a worse outcome in this population, resulting from a resistance to chemotherapy. The aim was the comparative assessment of angiogenesis in both CD5-positive and CD5-negative DLBCL, as well as in lymphatic tissues without lymphoproliferative diseases. The analysis included 36 cases of CD5-positive DLBCL (19 females and 17 males) aged 29-87 years (mean age 69), diagnosed and treated in the Maria Sklodowska-Curie Institute and Oncology Center and Medical University of Warsaw in 2002-2013. The control group comprised 28 cases of CD5-negative DLBCL (14 females and 14 males) aged 24-82 years (mean age 58.5). The secondary control group (13 cases) consisted of normal lymphatic tissue obtained from patients without lymphoproliferative diseases. The level of angiogenesis was assessed on the basis of immunohistochemical CD34, vWF and HIF1α expression measured using morphometric methods. CD5-positive DLBCL, in comparison to CD5-negative DLBCL, was characterized by: (1) higher mean of total blood vessel area, (2) higher mean total ratio of blood vessel area and staining intensity, (3) higher mean of total blood vessel area in regions defined as hot spots, (4) higher mean of total ratio of blood vessel area and staining intensity in hot spots. The measurements in lymph nodes without lymphoproliferative diseases lay between the values obtained in both DLBCL subgroups. We observed a significant exacerbation of angiogenesis in CD5-positive DLBCL in comparison to the CD5-negative subgroup, possibly explaining its more aggressive clinical course. Our data does not substantiate the hypothesis that angiogenesis is more pronounced in frequent CD5-negative DLBCL subgroup in comparison to benign lymphatic tissue.

Stimulatory effect of cobalt ions incorporated into calcium phosphate coatings on neovascularization in an in vivo intramuscular model in goats

Rapid vascularization of bone graft substitutes upon implantation is one of the most important challenges to overcome in order to achieve successful regeneration of large, critical-size bone defects. One strategy for stimulating vascularization during the regeneration process is to create a hypoxic microenvironment by either directly lowering the local oxygen tension, or by applying hypoxia-mimicking factors. Cells compensate for the hypoxic condition by releasing angiogenic factors leading to new blood vessel formation. In the present study, we explored the potential of cobalt ions (Co2+), known chemical mimickers of hypoxia, to stimulate vascularization within a bone graft substitute in vivo. To this end, Co2+ ions were incorporated into calcium phosphate (CaPs) coatings deposited on poly(lactic acid) (PLA) particles with their effect on the formation of new blood vessels studied upon intramuscular implantation in goats. PLA particles and CaP-coated particles without Co2+ ions served as controls. Pathological scoring of the inflammatory response following a 12-week implantation period showed no significant differences between the four types of materials. Based on histological and immunohistochemical analyses, both blood vessel area and number of blood vessels in CaP-coated PLA particles containing Co2+ were higher than in the uncoated PLA particles and CaP-coated PLA particles without Co2+. Analysis of blood vessel size distribution indicated abundant formation of small blood vessels in all the samples, while large blood vessels were predominantly found in PLA particles coated with CaP containing Co2+ ions. The results of this study support the use of CaPs containing Co2+ ions to enhance vascularization in vivo. Statement of SignificanceIn this work, we have investigated the potential of cobalt ions, incorporated into thin calcium phosphate (CaP) coatings that were deposited on particles of poly(lactic acid) (PLA), to induce neovascularization in vivo. Qualitative and quantitative histological and immunohistochemical analyses showed that both the number of blood vessels and the total blood vessel area were higher in CaP-coated PLA particles containing cobalt ions as compared to the uncoated PLA particles and CaP-coated PLA particles without the metallic additive. Furthermore, a wider distribution of blood vessel sizes, varying from very small to large vessels was specifically observed in samples containing cobalt ions.This in vivo study will significantly contribute to the existing knowledge on the use of bioinorganics, which are simple and inexpensive inorganic factors that can be used to control relevant biological process during tissue regeneration, such as vascularization. As such, we are convinced that this manuscript will be of interest to the readers of Acta Biomaterialia.

Distinctive Tooth‐Extraction Socket Healing: Bisphosphonate Versus Parathyroid Hormone Therapy

Patients with osteoporosis who receive tooth extractions are typically on either oral bisphosphonate or parathyroid hormone (PTH) therapy. Currently, the consequence of these therapies on hard- and soft-tissue healing in the oral cavity is not clearly defined. The aim of this study is to determine the differences in the therapeutic effect on tooth-extraction wound healing between bisphosphonate and PTH therapies. Maxillary second molars were extracted in Sprague Dawley rats (n = 30), and either bisphosphonate (zoledronate [Zol]), PTH, or saline (vehicle control [VC]) was administered for 10 days (n = 10 per group). Hard-tissue healing was evaluated by microcomputed tomography and histomorphometric analyses. Collagen, blood vessels, inflammatory cell infiltration, and cathepsin K expression were assessed in soft tissue using immunohistochemistry, quantitative polymerase chain reaction, and immunoblotting. Both therapies significantly increased bone fill and suppressed vertical bone loss. However, considerably more devital bone was observed in the sockets of rats on Zol versus VC. Although Zol increased the numbers of blood vessels, the total blood vessel area in soft tissue was significantly smaller than in VC. PTH therapy increased osteoblastic bone formation and suppressed osteoclasts. PTH therapy promoted soft-tissue maturation by suppressing inflammation and stimulating collagen deposition. Zoledronate therapy deters whereas PTH therapy promotes hard- and soft-tissue healing in the oral cavity, and both therapies prevent vertical bone loss.

Antiangiogenic Tumor Therapy by DNA Vaccine Inducing Aquaporin-1–Specific CTL Based on Ubiquitin–Proteasome System in Mice

Aquaporin-1 (AQP-1) is a water channel protein highly expressed in the vascular endothelial cells of proliferating tissues including malignant cancers. Given that in APC ubiquitinated peptides are effectively introduced into proteasomes from which CD8 epitopes are excised, we fused ubiquitin with AQP-1 (pUB-AQP-1) to produce a DNA vaccine. In C57BL/6J mice immunized with pUB-AQP-1, the growth of B16F10 melanoma was profoundly inhibited. The antitumor effect of the pUB-AQP-1 DNA vaccine was largely mediated by CD8 T cells, which secrete IFN-γ, perforin, and granzyme-B in the presence of APCs transfected with pUB-AQP-1. AQP-1-specific CD8 T cells possessed cytotoxic activity both in vivo and in vitro. After tumor challenge, the microvessel density decreased and the ratio of total blood vessel area to tumor area was significantly reduced as compared with control mice, resulting in a dramatic suppression of tumor growth. The immunization effect was completely abrogated in immunoproteasome-deficient mice. Strikingly this pUB-AQP-1 DNA vaccine was also effective against Colon 26 colon tumors (BALB/c) and MBT/2 bladder tumors (C3H/HeN). Thus, this ubiquitin-conjugated DNA immunization-targeting tumor vasculature is a valid and promising antitumor therapy. This vaccine works across the barriers of tumor species and MHC class I differences in host mice.

In ovo leptin administration inhibits chorioallantoic membrane angiogenesis in female chicken embryos through the STAT3-mediated vascular endothelial growth factor (VEGF) pathway

Previous studies indicate that leptin regulates placental angiogenesis and fetal growth in mammals and that in ovo leptin administration affects embryonic development and hatch weight in the chicken. To test the hypothesis that leptin affects embryonic growth through modifying chorioallantoic membrane (CAM) angiogenesis, we injected 0.5 μg of recombinant murine leptin into the albumen of fertilized eggs before incubation. On embryonic day 12 (E12), the number and the total area of blood vessels on CAM were measured, and expression of genes involved in angiogenesis was quantitated to show the possible mechanisms. Leptin in ovo administration decreased (P < 0.05) both the total area of blood vessels and the number of small-sized capillaries on CAM of E12 female chicken embryos, which coincided with significantly decreased (P < 0.05) embryo weight on E12 and BW at hatching. Vascular endothelial growth factor (VEGF) and inducible and endothelial nitric oxide synthases (iNOS and eNOS) were all downregulated (P < 0.05) in CAM both at the mRNA and protein/activity levels with reduced (P < 0.05) nitric oxide (NO) concentration in chorioallantoic fluid of female embryos. Furthermore, signal transducer and activator of transcription-3 (STAT3) was found to be diminished (P < 0.05) both at the mRNA and protein levels and associated with decreased (P < 0.05) binding of STAT3 to VEGF promotor in the CAM of leptin-treated E12 female embryos. These data suggest that in ovo leptin administration affects CAM angiogenesis and embryo growth in female chicken embryos, probably through STAT3-mediated VEGF/NO pathways.

Age-Related Decrease of the Retinal Vasculature Area Identified with a Novel Computer-Aided Analysis System

Fundus photographs enable non-invasive analysis of the status of the microcirculation by directly observing the retinal vasculature. Retinal microvascular abnormalities are important clinical markers of hypertension and arteriosclerosis, but retinal microvascular changes can be observed in older individuals without hypertension. In this study, our goal is to elucidate the effects of aging on fundus vessels in the retinal photograph. We analyzed retinal vessels of 161 healthy volunteers (49.5 ± 18.7 years, range 18-87) using in-house computer-aided measurement system to measure areas and diameters of all retinal vessels across the entire area of a retinal photograph. The vessels were segmented according to color, and then their area, size, length and thickness were measured by image processing. We also analyzed the correlation between total blood vessel area, age and mean arterial blood pressure (MABP). The decrease in total blood vessel area was dependent on both age and MABP. Moreover, decrease in blood vessel area was also correlated with age for the normotensive group. Furthermore, the slope of the regression line for retinal vessel area with MABP was significantly higher in participants aged ≤ 60 years than in those aged over 60 years. Changes in retinal vessel area with aging were observed in both arterioles and venules. In conclusion, we found the significant decrease in retinal vessel area that is correlated well with calendar age. Therefore, we need to carefully apply traditional classifications of fundus examination for hypertensive retinopathy in older individuals.

Different Etio-Pathogenesis in Intrauterine Growth Retardation in Developing Country: Histomorphometric Evidence

The present study was conducted to compare histomorphometric parameters of umbilical cord and its vessels in intrauterine growth retarded fetuses and normally grown fetuses. The study was conducted on umbilical cords taken from thirty intrauterine growth retarded (IUGR) fetuses and thirty normally grown fetuses (NGA). Histomorphometric parameters were measured with the help of planimeter. In the present study it was found that in cross section the total umbilical cord area in IUGR group is 59.00+3.67 mm2 and in the control group is 75.91+4.77mm2 (p<0.05). Jelly area in IUGR group is 47.90+3.11 mm2 and in control group is 60.26+4.55 mm2 (p<0.05). Total vessel area in control group is 15.45+1.73 mm2 and in IUGR group is 10.95±1.20mm2 (p <0.05). Total umbilical cord area, umbilical cord jelly area and total vessel area are significantly lower in the IUGR group but no significant difference was observed in total luminal area, total area of blood vessels and wall thickness of umbilical blood vessels. So in the study it was found that histomorphometric parameters of umbilical cord of IUGR fetuses are significantly lower than that of normally grown fetuses of similar gestational age.

Arteriogenesis: Noninvasive Quantification with Multi–Detector Row CT Angiography and Three-dimensional Volume Rendering in Rodents

To evaluate two-dimensional (2D) multi-detector row computed tomographic (CT) angiography and three-dimensional (3D) volume rendering for depiction of patterns of arterial growth and quantification of blood vessel density and volume. The institutional animal care and use committee approved this study. The right femoral artery and its branches were ligated and excised in 16 inbred Lewis rats; animals were randomly assigned to receive 70 microL Dulbecco's modified Eagle's medium (DMEM) or 1.5 x 10(7) bone marrow-derived mononuclear cells (BMC) from isogenic donor rats in 70 microL DMEM. At 2 weeks, CT angiography was performed with injection of 0.45 mL barium sulfate suspension at 0.7 mL/min, followed by silver staining. Number of blood vessels, area, mean area, volume, and blood vessel size distribution derived from digitally subtracted 2D CT angiographic sections were quantified; 3D images were reconstructed. Two-way analysis of variance and paired and unpaired Student t tests were performed. CT angiography showed two patterns of arterial growth: collateral arterial formation and branching arteriogenesis. Two-way analysis of variance indicated that differences within subjects (ischemic vs nonischemic legs) and between subjects (BMC vs DMEM treatment) were significant for total blood vessel area, total blood vessel volume, and mean of blood vessel area (P < .001). In the BMC group, there were significantly more arteries (mean, 241.6 +/- 77.0 [standard deviation] vs 196.4 +/- 75.2, P = .028), but mean cross-sectional area of these arteries was smaller in ischemic versus nonischemic legs (5.4 mm(2) +/- 1.2 vs 6.8 mm(2) +/- 1.3, P = .006). Total arterial area and volume did not differ significantly between ischemic and nonischemic legs. BMC injection had a substantial effect on arteriogenesis, with normalization of total arterial area and volume in the BMC group; this effect was successfully depicted.

Vascular changes in the rat brain during chronic hypoxia in the presence and absence of hypercapnia.

Changes in brain vascularity in adult rats during adaptation to chronic normobaric hypoxia with or without elevated CO(2) were morphometrically investigated. Immunohistochemistry with anti-rat endothelial cell antigen (RECA-1) antibody was carried out for the vascular analysis. After the rats were subjected to hypoxia for 2 to 8 weeks (wks)(10 percent O(2) in N(2)), the total area of blood vessels was measured in 6 brain regions. After 2 wks of hypoxia, the blood vessel area was found to be significantly increased in the frontal cortex, striatum, hippocampus, thalamus, cerebellum, and medulla oblongata, by 44% , 96% , 65% , 50% , 102% and 97% , respectively. The ratio of large vessels with an area > 500 micro m(2) was also increased in all brain regions. Hypoxic adaptation in brain vascularity did not change during 8 wks of hypoxia, and the hypoxia-induced levels measured in the vasculature returned to control levels 2 wks after the termination of hypoxia in areas of the brain other than the cortex and thalamus. In addition, hypoxia-induced changes in terms of the total vascular area and vessel size distribution were significantly inhibited by the elevation in CO(2), whereas chronic hypercapnia without hypoxia had no effect on brain vascularity. These findings suggested that adaptations in brain vascularity in response to hypoxia are rapidly induced, and there are regional differences in the reversibility of such vascular changes. Carbon dioxide is a potent suppressor of hypoxia-induced vascular changes, and may play an important role in vascular remodeling during the process of adaptation to chronic hypoxia.