Capillary Surface Area Research Articles

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement

The morphology of podocyte foot processes is obligatory for renal function. Here we describe a method for the superresolution-visualization of podocyte foot processes using structured illumination microscopy of the slit diaphragm, which before has only been achieved by electron microscopy. As a proof of principle, we measured a mean foot process width of 0.249 ± 0.068 µm in healthy kidneys and a significant higher mean foot process width of 0.675 ± 0.256 µm in minimal change disease patients indicating effacement of foot processes. We then hypothesized that the slit length per glomerular capillary surface area (slit diaphragm density) could be used as an equivalent for the diagnosis of effacement. Using custom-made software we measured a mean value of 3.10 ± 0.27 µm−1 in healthy subjects and 1.83 ± 0.49 µm−1 in the minimal change disease patients. As foot process width was highly correlated with slit diaphragm density (R2 = 0.91), we concluded that our approach is a valid method for the diagnosis of foot process effacement. In summary, we present a new technique to quantify podocyte damage, which combines superresolution microscopy with automatized image processing. Due to its diverse advantages, we propose this technique to be included into routine diagnostics of glomerular histopathology.

Effects of exercise on capillaries in the white matter of transgenic AD mice.

Previous studies have shown that exercise can prevent white matter atrophy in APP/PS1 transgenic Alzheimer’s disease (AD) mice. However, the mechanism of this protective effect remains unknown. To further understand this issue, we investigated the effects of exercise on the blood supply of white matter in transgenic AD mice. Six-month-old male APP/PS1 mice were randomly divided into a control group and a running group, and age-matched non-transgenic littermates were used as a wild-type control group. Mice in the running group ran on a treadmill at low intensity for four months. Then, spatial learning and memory abilities, white matter and white matter capillaries were examined in all mice. The 10-month-old AD mice exhibited deficits in cognitive function, and 4 months of exercise improved these deficits. The white matter volume and the total length, total volume and total surface area of the white matter capillaries were decreased in the 10-month-old AD mice, and 4 months of exercise dramatically delayed the changes in these parameters in the AD mice. Our results demonstrate that even low-intensity running exercise can improve spatial learning and memory abilities, delay white matter atrophy and protect white matter capillaries in early-stage AD mice. Protecting capillaries might be an important structural basis for the exercise-induced protection of the structural integrity of white matter in AD.

A composite smeared finite element for mass transport in capillary systems and biological tissue

One of the key processes in living organisms is mass transport occurring from blood vessels to tissues for supplying tissues with oxygen, nutrients, drugs, immune cells, and – in the reverse direction – transport of waste products of cell metabolism to blood vessels. The mass exchange from blood vessels to tissue and vice versa occurs through blood vessel walls. This vital process has been investigated experimentally over centuries, and also in the last decades by the use of computational methods. Due to geometrical and functional complexity and heterogeneity of capillary systems, it is however not feasible to model in silico individual capillaries (including transport through the walls and coupling to tissue) within whole organ models. Hence, there is a need for simplified and robust computational models that address mass transport in capillary–tissue systems. We here introduce a smeared modeling concept for gradient-driven mass transport and formulate a new composite smeared finite element (CSFE). The transport from capillary system is first smeared to continuous mass sources within tissue, under the assumption of uniform concentration within capillaries. Here, the fundamental relation between capillary surface area and volumetric fraction is derived as the basis for modeling transport through capillary walls. Further, we formulate the CSFE which relies on the transformation of the one-dimensional (1D) constitutive relations (for transport within capillaries) into the continuum form expressed by Darcy’s and diffusion tensors. The introduced CSFE is composed of two volumetric parts — capillary and tissue domains, and has four nodal degrees of freedom (DOF): pressure and concentration for each of the two domains. The domains are coupled by connectivity elements at each node. The fictitious connectivity elements take into account the surface area of capillary walls which belongs to each node, as well as the wall material properties (permeability and partitioning). The overall FE model contains geometrical and material characteristics of the entire capillary–tissue system, with physiologically measurable parameters assigned to each FE node within the model. The smeared concept is implemented into our implicit-iterative FE scheme and into FE package PAK. The first three examples illustrate accuracy of the CSFE element, while the liver and pancreas models demonstrate robustness of the introduced methodology and its applicability to real physiological conditions.

Impact of early mammalian development on the response of the O2 transport system to muscle contraction

A continuous supply of O2 to the cells in the body is necessary to maintain normal physiological function and the microcirculation is especially important in this matter as it is the site of O2 exchange. Under conditions of active/functional hyperemia following muscle contractions, the O2 transport system in older subjects does not appear to respond as rapidly and to the same degree as in younger subjects. With aging, elements of the O2 transport and regulatory systems are changed in such a way that matching O2 supply to O2 demand does not work as well in older as in younger subjects. An intravital microscopic approach is being used to show that developmental changes in the O2 transport system begin earlier than previously thought. Significant and rapid changes in the microvascular network of skeletal muscle have been observed during the first few weeks of postnatal development. Four different developmental groups of male Sprague‐Dawley rats (8, 16, 24, and 52 weeks) are used to investigate changes in the O2 demand component of the transport system by measuring oxygen consumption (VO2) and the PISFO2 dependence of VO2 under conditions of rest and contraction. VO2 was measured with a quasi‐continuous, flash‐synchronized, rapidly pressurizing airbag system to briefly arrest flow and determine the rate of change in oxygen tension (dPO2/dt). Changes in the O2 supply component are investigated by measuring capillary and interstitial fluid PO2 (PcapO2 and PISFO2 respectively, using phosphorescence quenching microscopy) and surface area available for O2 exchange (i.e., capillary density). Preliminary experiments were carried out on six animals that ranged from 8 w to 20 w in age with an average weight of 331 ± 35 g. Average baseline VO2 for the 8 w group was 307 ± 26 nL O2/cm3·s (n = 6), average baseline VO2 for the 16 w group was 287 ± 31 nL O2/cm3·s (n = 6), and average baseline VO2 for those in the 24 w group was 264 ± 12 nL O2/cm3·s (n = 6). Average baseline PISFO2 for the 8 w group was 68 ± 3 mmHg (n = 6), average baseline PISFO2 for the 16 w group was 74 ± 4 mmHg (n = 6), and average baseline PISFO2 for the 24 w group was 68 ± 3 mmHg (n = 6). The observed decreases in tissue VO2 are consistent with literature showing that whole body VO2 decreases from 499, 396, to 345 nL O2/cm3·s at 8 w, 16 w, and 24 w, respectively. However, it is still unclear how well matched O2 supply and demand are. As O2 demand increases by increasing muscle contraction, O2 supply should also increase to match it and PISFO2 is at least one element that links the two components of the transport system which requires further investigation.

Nephron morphometry in mice and rats using tomographic microscopy

The aim was to quantify the glomerular capillary surface area, the segmental tubular radius, length, and area of single nephrons in mouse and rat kidneys. Multiple 2.5-µm-thick serial Epon sections were obtained from three mouse and three rat kidneys for three-dimensional reconstruction of the nephron tubules. Micrographs were aligned for each kidney, and 359 nephrons were traced and their segments localized. Thirty mouse and thirty rat nephrons were selected for further investigation. The luminal radius of each segment was determined by two methods. The luminal surface area was estimated from the radius and length of each segment. High-resolution micrographs were recorded for five rat glomeruli, and the capillary surface area determined. The capillary volume and surface area were corrected for glomerular shrinkage. A positive correlation was found between glomerular capillary area and proximal tubule area. The thickest part of the nephron, i.e., the proximal tubule, was followed by the thinnest part of the nephron, i.e., the descending thin limb, and the diameters of the seven identified nephron segments share the same rank in the two species. The radius and length measurements from mouse and rat nephrons generally share the same pattern; rat tubular radius-to-mouse tubular radius ratio ≈ 1.47, and rat tubular length-to-mouse tubular length ratio ≈ 2.29, suggesting relatively longer tubules in the rat. The detailed tables of mouse and rat glomerular capillary area and segmental radius, length, and area values may be used to enhance understanding of the associated physiology, including existing steady-state models of the urine-concentrating mechanism.

Effects of preterm birth and ventilation on glomerular capillary growth in the neonatal lamb kidney.

Preterm birth is linked to the development of hypertension later in life. This may relate to impaired glomerular capillary growth following preterm birth. The aim of this study was to determine the effects of preterm birth, and/or ventilation, on glomerular capillary growth in the neonatal lamb kidney. Four experimental groups were analysed: preterm lambs delivered at 130 days gestation (term = 147 days) and mechanically ventilated for 3 days (preterm ventilated: n = 9), 133 days gestational controls (gestational control: n = 5), term controls, unassisted breathing for 3 days (term control: n = 8), and term lambs ventilated for 3 days (term ventilated: n = 5). In perfusion-fixed kidneys, total nephron number, average total capillary length, and surface area per renal corpuscle were stereologically assessed, and total renal filtration surface area (TRFSA) was calculated. In comparison with term controls, preterm lambs had significantly reduced glomerular capillary length, surface area, and TRFSA, indicative of a low renal functional capacity. Term-ventilated lambs exhibited significantly reduced glomerular capillary length and surface area compared with term controls, indicating that ventilation impairs glomerular capillary growth independently of preterm birth. Impaired glomerular capillary growth and subsequent reduced TRFSA following preterm birth may mediate the increased predisposition to hypertension later in life.

Endothelium-Derived Hyperpolarizing Factors: A Potential Therapeutic Target for Vascular Dysfunction in Obesity and Insulin Resistance

The endothelium consists of a single layer of cells that serves as a barrier between blood and tissues and actively participates in the regulation of vascular tone and function (1). The influence of the endothelium on blood flow in arterioles and capillaries is modulated by the synthesis and release of a number of endothelial-derived relaxing and constricting substances such as nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factors (EDHF) (2). However, the balance or homeostasis between endothelial relaxing and constricting factors is disrupted in insulin-resistant states including obesity, type 2 diabetes mellitus (T2DM), and hypertension, all of which promote cardiovascular disease (CVD) (1,2). For example, endothelial cell (EC) dysfunction is caused by impairment of endothelial-dependent vasodilation in various vascular beds such as skeletal muscle arterioles and capillaries. Both conduit arteries and microvessels are very important for insulin and glucose metabolism as an increase in capillary surface area prompts both insulin and glucose delivery and subsequent glucose uptake in skeletal muscle tissue (3,4). In healthy individuals, systemic and local infusion of insulin induces NO production to prompt insulin-mediated capillary recruitment and microvascular blood flow, resulting in an increase in forearm blood volume (2). One study demonstrated that endothelial NO synthase inhibition significantly blunted the insulin-stimulated increase of ultrasound-assessed femoral blood flow without any changes in blood pressure and heart rate in rats (5). In parallel, insulin-stimulated capillary recruitment and glucose uptake were completely abolished (5), suggesting that local capillary NO bioavailability plays a key role in the regulation of insulin-stimulated skeletal muscle capillary recruitment and glucose uptake. The …

Tumor Angiogenesis – factors influencing the development of a tumor vascular network and assessment of neoangiogenesis in histopathological samples

Angiogenesis involves the formation of capillaries on the basis of already existing blood vessels. It is a multi-stage process resultant from the combined influence of pro- and anti-angiogenic factors. It begins with the stimulation of endothelial cells and degeneration of the basilemma and extracellular matrix, followed by the proliferation of endothelial cells and eventually the formation of a new vessel. The final stage involves a synthesis of the vessel's basilemma and incorporation of pericytes stabilizing the capillary tube. Any study of angiogenesis necessitates a direct assessment of tissue vascularisation and indirect assessment of the occurrence of pro-angiogenic factors. The degree of vascularisation is determined on the basis of the number of capillaries and endothelial cell concentrations per a surface unit of a given tissue or organ. The same is achieved by staining histopathological samples with immunohistochemical methods using panendothelial antibodies: anti FVIII, anti-CD31 and anti CD34. The antibodies identifying proliferating endotelial cells are also specific to the evaluation of neoangiogenesis. The same include monoclonal antibodies: E9 and TEC-11. The quantitative analysis of blood vessel density in tumor tissue (MVD - microvessel density) is performed with the Weidner method. The total microvascular area (TVA) is also considered a significant angiogenic factor. The assessment of the relative surface area of capillaries within a tumor is performed using Chalkley's method, vessel branching count (VBC) and angiogenic index. Indirect methods of angiogenesis assessment involve the determination of angiogenic cytokine production and the expression of their cellular receptors. The study of modulators and assessment of angiogenesis may facilitate more efficient tumor diagnostics and therapy.

Estimation of the number of alveolar capillaries by the Euler number (Euler-Poincaré characteristic).

The lung parenchyma provides a maximal surface area of blood-containing capillaries that are in close contact with a large surface area of the air-containing alveoli. Volume and surface area of capillaries are the classic stereological parameters to characterize the alveolar capillary network (ACN) and have provided essential structure-function information of the lung. When loss (rarefaction) or gain (angiogenesis) of capillaries occurs, these parameters may not be sufficient to provide mechanistic insight. Therefore, it would be desirable to estimate the number of capillaries, as it contains more distinct and mechanistically oriented information. Here, we present a new stereological method to estimate the number of capillary loops in the ACN. One advantage of this method is that it is independent of the shape, size, or distribution of the capillaries. We used consecutive, 1 μm-thick sections from epoxy resin-embedded material as a physical disector. The Euler-Poincaré characteristic of capillary networks can be estimated by counting the easily recognizable topological constellations of "islands," "bridges," and "holes." The total number of capillary loops in the ACN can then be calculated from the Euler-Poincaré characteristic. With the use of the established estimator of alveolar number, it is possible to obtain the mean number of capillary loops per alveolus. In conclusion, estimation of alveolar capillaries by design-based stereology is an efficient and unbiased method to characterize the ACN and may be particularly useful for studies on emphysema, pulmonary hypertension, or lung development.

The form-stable phase change materials based on polyethylene glycol and functionalized carbon nanotubes for heat storage

Form-stable phase change materials (PCMs) for heat storage based on polyethylene glycol (PEG) and functionalized multi-wall carbon nanotubes (MWNTs) were prepared via a blending and impregnating method. The structural and thermal properties of the as-prepared PCMs were analyzed by various techniques. The phase change temperatures and enthalpies of the composite PCMs with hydroxylated (-OH), carboxylated (-COOH) and aminoated (-NH2) MWNTs (PEG/MWNT-x PCMs) are lower than those with pristine MWNTs (PEG/MWNT PCMs). The influence of functional groups on the phase change behavior of the composite PCMs follows the order of: MWNT-COOH > MWNT-NH2 > MWNT-OH > MWNT. Capillary forces, surface areas and hydrogen bonding are mainly influential on the phase change behavior of PEG/MWNT-x PCMs.

Contrast-Enhanced Ultrasound Assessment of Impaired Adipose Tissue and Muscle Perfusion in Insulin-Resistant Mice

In diabetes mellitus, reduced perfusion and capillary surface area in skeletal muscle, which is a major glucose storage site, contribute to abnormal glucose homeostasis. Using contrast-enhanced ultrasound, we investigated whether abdominal adipose tissue perfusion is abnormal in insulin resistance and correlates with glycemic control. Contrast-enhanced ultrasound perfusion imaging of abdominal adipose tissue and skeletal muscle was performed in obese insulin resistance (db/db) mice at 11 to 12 or 14 to 16 weeks of age and in control lean mice. Time-intensity data were analyzed to quantify microvascular blood flow (MBF) and capillary blood volume (CBV). Blood glucose response for 1 hour was measured after insulin challenge (1 U/kg, IP). Compared with control mice, db/db mice at 11 to 12 and 14 to 16 weeks had a higher glucose concentration area under the curve after insulin (11.8±2.8, 20.6±4.3, and 28.4±5.9 mg·min/dL [×1000], respectively; P=0.0002) and also had lower adipose MBF (0.094±0.038, 0.035±0.010, and 0.023±0.01 mL/min per gram; P=0.0002) and CBV (1.6±0.6, 1.0±0.3, and 0.5±0.1 mL/100 g; P=0.0017). The glucose area under the curve correlated in a nonlinear fashion with both adipose and skeletal muscle MBF and CBV. There were significant linear correlations between adipose and muscle MBF (r=0.81) and CBV (r=0.66). Adipocyte cell volume on histology was 25-fold higher in 14- to 16-week db/db versus control mice. Abnormal adipose MBF and CBV in insulin resistance can be detected by contrast-enhanced ultrasound and correlates with the degree of impairment in glucose storage. Abnormalities in adipose tissue and muscle seem to be coupled. Impaired adipose tissue perfusion is in part explained by an increase in adipocyte size without proportional vascular response.

Mathematical model of renal hemodynamic and structural consequences of increased proximal sodium reabsorption in early diabetic kidney disease

A mathematical model of integrative renal and cardiovascular function was used to evaluate the potential role of increased sodium reabsorption through sodium glucose cotransporters (SGLT) in the proximal tubule (PT) in driving hemodynamic and structural changes in the kidney in early diabetic kidney disease. First, the effect of glomerular pressure (GP) on adaptive hypertrophy of the glomerulus was modeled based on literature data from subtotal nephrectomy studies. Increased GP following nephrectomy was assumed to drive glomerular hypertrophy, increasing capillary surface area and thus the ultrafiltration coefficient (Kf). As Kf increased, GP returned to close to normal levels, and blood pressure and glomerular filtration rate (GFR) were maintained near pre‐nephrectomy levels. This effect of GP on hypertrophy was assumed to be a general adaptive mechanism, not specific to nephrectomy. Next, increased sodium reabsorption through SGLT resulting from high glucose levels in diabetes was modeled as an increase in the rate of PT sodium reabsorption. This resulted in an initial elevation in both GP and GFR through tubuloglomerular and neurohormonal feedback mechanisms. Over time, Kf increased while GP returned toward baseline. However, maximum hypertrophy occurred before GP completely normalized, indicating that SGLT‐driven PT sodium reabsorption can lead to sustained elevations in GP. This sustained increase in GP, as well as repeated spikes in glucose that elevate GP further, may cause cumulative damage to the glomerular membrane, leading to the progressive decline in GFR that follows the hyperfiltration phase in diabetic kidney disease. An interactive interface to the model is being developed and is available at qsp.engr.uga.edu:3838/CVRmod.

Effects of long-term exercise on spatial learning, memory ability, and cortical capillaries in aged rats.

BackgroundThis study aimed to determine the effects of long-term running exercise on spatial learning, spatial memory, and cortical capillaries in aged rats.Material/MethodsFourteen-month-old female and male Sprague-Dawley rats were randomly divided into an exercised group (EG) and a non-exercised group (NG). The EG rats were trained on treadmill running for 4 or 14 months. The NG rats were housed under identical conditions without running. Spatial learning and memory were assessed with the Morris water maze. The cortical capillary parameters were quantitatively investigated using immunohistochemical and stereological methods.ResultsThe escaped latencies of the EG were significantly different from those of the NG in 18-month-old females and 28-month-old males (p<0.05). However, 28-month-old females and 18-month-old males showed no differences in escape latency between the EG and NG (p>0.05). In 28-month-old female rats, stereological techniques showed significant differences between the EG and NG in the cortical capillary volume (median, 22.55 vs. 11.42, p<0.05) and the cortical capillary surface area (median, 7474.13 vs. 3935.90, p<0.05). In 28-month-old male rats, the EG had a significantly longer total cortical capillary length (median, 530.35 vs. 156.27, p<0.05), significantly larger cortical capillary volume (median, 16.47 vs. 3.65, p<0.01), and a significantly larger cortical capillary total surface area (median, 7885.79 vs. 1957.16, p<0.01) compared with the NG group.ConclusionsThese data demonstrate that exercise improved spatial learning, memory capacity and cortical capillaries in aged rats.

Possibilities of differentiation of solitary focal liver lesions by computed tomography perfusion.

To evaluate possibilities of computed tomography (CT) perfusion in differentiation of solitary focal liver lesions based on their characteristic vascularization through perfusion parameters analysis. Prospective study was conducted on 50 patients in the period 2009-2012. Patients were divided in two groups: benign and malignant lesions. The following CT perfusion parameters were analyzed: blood flow (BF), blood volume (BV), mean transit time (MTT), capillary permeability surface area product (PS), hepatic arterial fraction (HAF), and impulse residual function (IRF). During the study another perfusion parameter was analyzed: hepatic perfusion index (HPI). All patients were examined on Multidetector 64-slice CT machine (GE) with application of perfusion protocol for liver with i.v. administration of contrast agent. In both groups an increase of vascularization and arterial blood flow was noticed, but there was no significant statistical difference between any of 6 analyzed parameters. Hepatic perfusion index values were increased in all lesions in comparison with normal liver parenchyma. Computed tomography perfusion in our study did not allow differentiation of benign and malignant liver lesions based on analysis of functional perfusion parameters. Hepatic perfusion index should be investigated in further studies as a parameter for detection of possible presence of micro-metastases in visually homogeneous liver in cases with no lesions found during standard CT protocol.

Investigation of concrete exposed to dual sulfate attack

Durability of concrete exposed to sulfates has primarily been studied on specimens fully-submerged in sulfate solutions. However, field experience shows that concrete exposed to sulfates can suffer from surface scaling above ground level due to physical attack. This damage has often been ignored and even confused with chemical sulfate attack. In this study, concrete partially-immersed in sulfate solutions and exposed to cyclic temperature and relative humidity was explored. Results show that concrete can experience dual sulfate attack. The lower immersed portion can suffer from chemical sulfate attack, while the upper portion can be vulnerable to physical attack. Lowering the water-to-binder ratio and moist-curing reduced surface scaling above the solution level, since the volume of pores was decreased. Although partial replacement of cement with pozzolans also decreased the pore volume, surface scaling increased due to increased proportion of small diameter pores and associated growth of capillary suction and surface area for evaporation.

Abstract 354: Inducible Disruption of Endothelial Hyaluronan Synthase-2 Expression in Adult Mice Reveals a Direct Involvement of Glomerular Endothelial Surface Hyaluronan in Development of Albuminuria and Renal Injury

We demonstrated that infusion with hyaluronidase of C57Bl/6 mice for 4 wks to chronically reduce EC surface hyaluronan expression resulted in glomerular albumin passage and podocyte injury. However, with systemic hyaluronan degradation it precludes definitive conclusions on cellular-specific effects. Here we investigated the hypothesis that it is specifically the structure and integrity of the glomerular EC glycocalyx that determines whether or not albumin passage across the capillary wall occurs, and subsequently protects against development of kidney injury. To this end, we generated a conditional EC specific hyaluronan synthase2-knock out (HAS2–KO) mouse model. 8 Week old B6.VECad-creERT2.has2f/f.Rosa26-tdTomato were injected i.p. for 5 consecutive days with 2mg/0.2μL tamoxifen (control B6.VECad-creERT2.Rosa26-tdTomato) to induce an endothelial specific deletion of has2. Compared to ctrl, these mice show that 4 wks after induction, weight gain was significantly more increased (mean ± SEM; 2.9 ± 0.7 [n=8] vs. 5.2 ± 0.6g [n=11], p=0.02). With prominent endogenous albumin passage over the barrier, visualized as a DAB reaction product in electron microscopy after anti-albumin-HRP antibody incubation of 2%PFA fixated tissue slides, visible 2 wks after induction, a significant (p=0.036) increased ACR (36.0 ± 1.9 [n=8] vs. 57.7 ± 7.9 [n=11]) was found only at wk 4. During this period no significant change in BP (85 ± 2/114 ± 2 [n=8] vs. 90 ± 4/119 ± 4mmHg [n=10], diastolic/systolic) or in heart rate (712 ± 14 [n=8] vs. 747 ± 8beats/min [n=10]) was observed. Renal morphology revealed with comparable glomerular surface area (5342 ± 270 [n=3] vs. 5508 ± 250 μm2 [n=6]), significant increase of glomerular mesangial area (20.1 ± 0.9 [n=3]) vs. 38.4 ± 1.2% [n=6], p&lt;0.01) and capillary surface area (16.6 ± 1.0 [n=3]) vs. 22.8 ± 0.7% [n=6], p=0.046). In conclusion, the data so far indicate that an intact glomerular EC-ESL is essential for glomerular integrity and prevention of albumin filtration is Key to preserving local quiescence of mesangial cells and podocytes. The conditional EC specific HAS2–KO mouse seems to be a valid model to assess the role of ESL in glomerular permeability barrier and vascular induced glomerular changes.

How the Kidney Is Impacted by the Perinatal Maternal Environment to Develop Hypertension1

Environmental conditions during perinatal development such as maternal undernutrition, maternal glucocorticoids, placental insufficiency, and maternal sodium overload can program changes in renal Na(+) excretion leading to hypertension. Experimental studies indicate that fetal exposure to an adverse maternal environment may reduce glomerular filtration rate by decreasing the surface area of the glomerular capillaries. Moreover, fetal responses to environmental insults during early life that contribute to the development of hypertension may include increased expression of tubular apical or basolateral membrane Na(+) transporters and increased production of renal superoxide leading to enhanced Na(+) reabsorption. This review will address the role of these potential renal mechanisms in the fetal programming of hypertension in experimental models induced by maternal undernutrition, fetal exposure to glucocorticoids, placental insufficiency, and maternal sodium overload in the rat.

Increased Angiogenic Property Of Human Peripheral Blood Monocytes By ex Vivo Culture With c-Mpl Agonists In Hindlimb Ischemia Mouse Model

Human peripheral blood mononuclear cells (PB-MNCs) include some populations which have angiogenic properties. Pro-angiogenic monocytes from PB-MNCs are considered as one of candidates for angiogenic therapy in regenerative medicine. Indeed, in a recent German clinical post-infarction remodeling study (TOPCARE-AMI) for ischemic heart disease, the ex vivo culture of PB-MNCs was employed. However, in this trial, there were different therapeutic efficacies in each case, possibly due to the different expansion efficacy of the ex vivo culture of PB-MNCs using autologous serum.In order to resolve this issue, we developed a new serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Floating spheres obtained by this serum-free culture system were mainly composed of CD11b+ monocytes. Interestingly, the mRNA expression of c-Mpl (thrombopoietin receptor) was markedly elevated compared with PB-MNCs, suggesting c-Mpl agonist could increase angiogenic property of cultured CD11b+ monocytes. Therefore, we assessed the impact of c-Mpl agonists on PB-MNC cultures in our serum-free method composed of X-VIVO15 medium with VEGF and bFGF.Both recombinant human thrombopoietin (rHuTPO) and romiplostim, a clinical grade second-generation TPO-receptor agonist, successfully increased sphere formations regarding both the number and size. The expressions of angiogenic factors, IL-8, CXCR4, and vasohibin-2, mRNA of CD11b+ monocytes cultured with c-Mpl agonists were up-regulated, indicating that cultivated CD11b+ monocytes have a proangiogenic potential.Finally, we investigated the proangiogenic potential of PB-MNCs derived CD11b+ monocytes in a hindlimb ischemia model utilizing BALB/c nude mice. Mice were randomly assigned to 7 groups: control mouse group (PBS-injected), freshly isolated CD11b+ monocyte-injected mouse group, cultivated CD11b+ monocyte with 2ng/ml and 20ng/ml rHuTPO -injected mouse groups, cultivated CD11b+ monocyte with 100ng/ml and 1000ng/ml romiplostim -injected mouse groups, cultivated CD11b+ monocyte without rHuTPO and romiplostim -injected mouse group. The intramuscular injection of CD11b+ monocytes cultivated with 20 ng/ml rHuTPO into the ischemic limb completely rescued the limbs from auto-amputations or foot necrosis, while only one (10.0%) of the control mice could be rescued. In addition, the intramuscular injection of both freshly isolated CD11b+ monocytes and CD11b+ monocytes cultivated without rHuTPO and romiplostim had a weak rescue effect on the ischemic limbs (8 and 7 of 10 mice had auto-amputations or foot necrosis, respectively). The salvage rate from necrosis in cultivated CD11b+ monocyte with romiplostim-injected mouse group is also superior to that in cultivated CD11b+ monocyte without rHuTPO-injected mouse group. Analysis of blood perfusion by a laser Doppler perfusion imaging system showed a significantly higher recovery in mice receiving the CD11b+ monocytes cultivated with 2 ng/ml or 20 ng/ml rHuTPO or 100ng/ml romiplostim 1 week after surgery. The functional capillary density and surface area visualized by perfusion with BS-I lectin also significantly increased in the rHuTPO- or romiplostim-treated group.In conclusion, an ex vivo addition of c-Mpl agonists augmented the pro-angiogenic activity of peripheral CD11b+ monocytes, and this method would be promising for human cell therapy to induce vascular regeneration by activating the angiogenic property in human peripheral blood-derived monocytes. Disclosures:Mizukami:The New Energy and Industrial Technology Development Organization of Japan: Research Funding.

Increased microvascular proliferation is negatively correlated to tumour blood flow and is associated with unfavourable outcome in endometrial carcinomas

Background:We aimed to study the angiogenic profile based on histomorphological markers in endometrial carcinomas in relation to imaging parameters obtained from preoperative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion-weighted imaging (DWI) and to explore the potential value of these markers to identify patients with poor outcome.Methods:In fifty-four surgically staged endometrial carcinoma patients, immunohistochemical staining with factor VIII and Ki67 allowed assessment of microvessel density (MVD) and microvascular proliferation reflecting tumour angiogenesis. In the same patients, preoperative pelvic DCE-MRI and DWI allowed the calculation of parameters describing tumour microvasculature and microstructure in vivo.Results:Microvascular proliferation was negatively correlated to tumour blood flow (Fb) (r=−0.36, P=0.008), capillary permeability surface area product (PS) (r=−0.39, P=0.004) and transfer from the blood to extravascular extracellular space (EES) (Ktrans) (r=−0.40, P=0.003), and was positively correlated to tumour volume (r=0.34; P=0.004). High-tumour microvascular proliferation, low Fb and low Ktrans were all significantly associated with reduced progression/recurrence-free survival (P<0.05).Conclusion:Disorganised angiogenesis with coexisting microvascular proliferation and low tumour blood flow is a poor prognostic factor supporting that hypoxia is associated with progression and metastatic spread in endometrial carcinomas.