Fluorescent microangiography is a novel and widely applicable technique for delineating the renal microvasculature.

Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 µm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections.

Culture-modified bone marrow cells attenuate cardiac and renal injury in a chronic kidney disease rat model via a novel antifibrotic mechanism.

BACKGROUND: Most forms of chronic kidney disease are characterized by progressive renal and cardiac fibrosis leading to dysfunction. Preliminary evidence suggests that various bone marrow-derived cell populations have antifibrotic effects. In exploring the therapeutic potential of bone marrow derived cells in chronic cardio-renal disease, we examined the anti-fibrotic effects of bone marrow-derived culture modified cells (CMCs) and stromal cells (SCs). METHODOLOGY/PRINCIPAL FINDINGS: In vitro, CMC-conditioned medium, but not SC-conditioned medium, inhibited fibroblast collagen production and cell signalling in response to transforming growth factor-beta. The antifibrotic effects of CMCs and SCs were then evaluated in the 5/6 nephrectomy model of chronic cardio-renal disease. While intravascular infusion of 10(6) SCs had no effect, 10(6) CMCs reduced renal fibrosis compared to saline in the glomeruli (glomerulosclerosis index: 0.8+/-0.1 v 1.9+/-0.2 arbitrary units) and the tubulointersitium (% area type IV collagen: 1.2+/-0.3 v 8.4+/-2.0, p<0.05 for both). Similarly, 10(6) CMCs reduced cardiac fibrosis compared to saline (% area stained with picrosirius red: 3.2+/-0.3 v 5.1+/-0.4, p<0.05), whereas 10(6) SCs had no effect. Structural changes induced by CMC therapy were accompanied by improved function, as reflected by reductions in plasma creatinine (58+/-3 v 81+/-11 micromol/L), urinary protein excretion (9x/divided by 1 v 64x/divided by 1 mg/day), and diastolic cardiac stiffness (left ventricular end-diastolic pressure-volume relationship: 0.030+/-0.003 v 0.058+/-0.011 mm Hg/microL, p<0.05 for all). Despite substantial improvements in structure and function, only rare CMCs were present in the kidney and heart, whereas abundant CMCs were detected in the liver and spleen. CONCLUSIONS/SIGNIFICANCE: Together, these findings provide the first evidence suggesting that CMCs, but not SCs, exert a protective action in cardio-renal disease and that these effects may be mediated by the secretion of diffusible anti-fibrotic factor(s).

The (Pro)renin receptor: site-specific and functional linkage to the vacuolar H+-ATPase in the kidney.

The (pro)renin receptor ([P]RR) is a transmembrane protein that binds both renin and prorenin with high affinity, increasing the catalytic cleavage of angiotensinogen and signaling intracellularly through mitogen-activated protein kinase activation. Although initially reported as having no homology with any known membrane protein, other studies have suggested that the (P)RR is an accessory protein, named ATP6ap2, that associates with the vacuolar H(+)-ATPase, a key mediator of final urinary acidification. Using in situ hybridization, immunohistochemistry, and electron microscopy, together with serial sections stained with nephron segment-specific markers, we found that (P)RR mRNA and protein were predominantly expressed in collecting ducts and in the distal nephron. Within collecting ducts, the (P)RR was most abundant in microvilli at the apical surface of A-type intercalated cells. Dual-staining immunofluorescence demonstrated colocalization of the (P)RR with the B1/2 subunit of the vacuolar H(+)-ATPase, the ion exchanger that secretes H(+) ions into the urinary space and that associates with an accessory subunit homologous to the (P)RR. In collecting duct/distal tubule lineage Madin-Darby canine kidney cells, extracellular signal-regulated kinase 1/2 phosphorylation, induced by either renin or prorenin, was attenuated by the selective vacuolar H(+)-ATPase inhibitor bafilomycin. The predominant expression of the (P)RR at the apex of acid-secreting cells in the collecting duct, along with its colocalization and homology with an accessory protein of the vacuolar H(+)-ATPase, suggests that the (P)RR may function primarily in distal nephron H(+) transport, recently noted to be, at least in part, an angiotensin II-dependent phenomenon.

Natural history of experimental arterial chronic total occlusions.

OBJECTIVES: We sought to perform the first systematic study of the natural history of chronic total arterial occlusions (CTOs) in an experimental model. BACKGROUND: Angioplasty of CTOs has low success rates. The structural and perfusion changes during CTO maturation, which may adversely affect angioplasty outcome, have not been systematically studied. METHODS: Occlusions were created in 63 rabbit femoral arteries by thrombin injection. Histology, contrast-enhanced magnetic resonance imaging, relative blood volume (RBV) index, and micro-computed tomography imaging were analyzed at 2, 6, 12, and 18 to 24 weeks. RESULTS: Early changes were characterized by an acute inflammatory response and negative arterial remodeling, with >70% reduction of arterial cross-sectional area (CSA) from 2 to 6 weeks. Intraluminal neovascularization of the CTO occurred with a 2-fold increase in total (media + intima) microvessel CSA from 2 to 6 weeks (0.014 +/- 0.002 mm2 to 0.023 +/- 0.005 mm2, p = 0.0008) and a 3-fold increase in RBV index (5.1 +/- 1.9% to 16.9 +/- 2.7%, p = 0.0008). However at later time periods, there were significant reductions in both RBV (3.5 +/- 1.1%, p < 0.0001) and total microvessel CSA (0.017 +/- 0.002 mm2, p = 0.011). Micro-computed tomography imaging demonstrated a corkscrew-like recanalization channel at the proximal end at 6 weeks that regressed at later time points. These vascular changes were accompanied by a marked decrease in proteoglycans and accumulation of a collagen-enriched extracellular matrix, particularly at the entrance ("proximal fibrous cap"). CONCLUSIONS: This study is the first to systematically analyze compositional changes occurring during CTO maturation, which may underlie angioplasty failure. Negative remodeling, regression of intraluminal channels, and CTO perfusion, together with the accumulation of dense collagen, may represent important targets for novel therapeutic interventions.

Simultaneous right atrioventricular pacing: a novel model to study atrial remodeling and fibrillation in the setting of heart failure.

BACKGROUND: Atrial fibrillation (AF) is a common arrhythmia which contributes to morbidity and mortality in patients with heart failure (HF). Atrial remodeling is a key substrate for the development of AF in HF. However, experimental models that study AF in the setting of HF have important limitations. We evaluated a new dog model of atrial remodeling and AF. METHODS AND RESULTS: Twenty-two mongrel dogs were randomized into 2 groups: 14 dogs with simultaneous atrioventricular pacing (SAVP) for 2 weeks (220 beats/min, no AV delay) and 8 control dogs with no pacing. SAVP for 2 weeks induced marked changes in atrial mechanical function and conduction. Left atrial area fractional shortening decreased 61 +/- 17%, whereas left ventricular area fractional shortening decreased by 38 +/- 18% from baseline (both P < .05). Conduction slowed and conduction heterogeneity increased. AF was induced in 83% of SAVP dogs, lasting a median of 1600 seconds, versus no dogs with induced AF in the controls. SAVP significantly increased nonfibrillar collagen in the mid-myocardium of both atrial appendages and matrix metalloproteinase-9 activity. CONCLUSIONS: SAVP in dogs induces structural and electrical remodelling that form the substrate for reproducibly inducible AF. This novel model may be useful for studies of the pathophysiology and treatment of AF in heart failure.

Experimental studies of atrial fibrillation: a comparison of two pacing models.

Rapid ventricular pacing (RVP) is a well-established animal model of atrial fibrillation (AF). However, this model is limited by a high mortality rate and severe heart failure. The purpose of our study was to assess a new canine model of inducible AF. We performed acute, short-term, simultaneous atrioventricular pacing (SAVP) and RVP (in random order) in 14 dogs for 30 s. SAVP produced more echocardiographic pulmonary venous flow reversal, a greater increase in mean pulmonary capillary wedge pressure, and a significantly greater decrease in left atrial emptying function (-84.4 +/- 38.6% vs. -23.7 +/- 27.1%, P < 0.05) than RVP. Thirty dogs were randomized to three, longer-term, study groups: eight dogs in the control group (no pacing), eight dogs in the RVP group (2 wk at 240 beats/min followed by 3 wk at 220 beats/min), and fourteen dogs in the SAVP group (2 wk at 220 beats/min). SAVP induced less left ventricular dysfunction but more left atrial dysfunction than RVP. SAVP dogs had similar atrial effective refractory periods as RVP dogs but more heterogeneity in conduction and more AF inducibility (83% vs. 40%, P < 0.05) and maintenance (median 1,660 vs. 710 s, P < 0.05) than RVP dogs. SAVP induced more collagen turnover and was associated with a significantly greater increase in type III collagen in the atria compared with RVP dogs (6.9 +/- 1.5 vs. 4.8 +/- 1.6, respectively, P < 0.05 vs. 1.1 +/- 0.7 in unpaced control dogs). In conclusion, the SAVP model induced profound mechanical and substrate atrial remodeling and reproducible sustained AF. This new model is clinically relevant and may be useful for testing AF interventions.

Long chain n-3 polyunsaturated fatty acids reduce atrial vulnerability in a novel canine pacing model.

AIM: Our objective was to assess the effect of omega-3 polyunsaturated fatty acids (n-3 PUFAs) on atrial fibrillation (AF) vulnerability and atrial structure in a new model of atrial cardiomyopathy. METHODS AND RESULTS: Dogs were studied in three groups: seven control dogs (UNPACED) and 24 dogs undergoing simultaneous atrioventricular pacing (for 2 weeks) assigned to placebo treatment (SAVP-PLACEBO, n = 12 dogs) or oral n-3 PUFAs (1 g/day) treatment (SAVP-PUFA, n = 12 dogs). SAVP-PUFA dogs had less AF inducibility (percentage of burst attempts leading to AF episodes: 5.5 +/- 7.4 vs. 20.4 +/- 14.2, P < 0.001) and maintenance [median AF duration: 601 s (377-1216) vs. 1598 s (1195-2400), P < 0.05] than SAVP-PLACEBO dogs. SAVP-PUFA dogs had significantly less local slowing of conduction and conduction heterogeneity than SAVP-PLACEBO dogs. SAVP-PUFA dogs had a significantly smaller increase in atrial matrix metalloproteinase-9 activity and in collagen type I and III messenger RNA expression (in arbitrary units) than SAVP-PLACEBO dogs (0.62 +/- 0.51 vs. 10.80 +/- 5.61, respectively for collagen I, P < 0.05; 1.66 +/- 0.48 vs. 5.24 +/- 1.16, respectively, for collagen III, P < 0.05). CONCLUSION: n-3 PUFA supplementation can reduce AF vulnerability in a new canine pacing model of atrial cardiomyopathy. The mechanism may be related to attenuation of collagen turnover.

Microvascular regeneration in established pulmonary hypertension by angiogenic gene transfer.

Pulmonary arterial hypertension (PAH) is characterized by widespread loss of pulmonary microvasculature. Therefore we hypothesized that angiogenic gene therapy would reverse established PAH, in part restoring the lung microcirculation. Three weeks after monocrotaline (MCT) treatment, Fisher 344 rats were randomized to receive a total of either 1.5 x 10(6) syngeneic fibroblasts (FB) transfected with vascular endothelial growth factor A (VEGF), endothelial NO synthase (eNOS), or null-plasmid transfected FBs. Right ventricular systolic pressure (RVSP) was similarly increased in all MCT-treated groups at the time of gene transfer. Animals receiving the null-vector progressed to severe PAH by Day 35 (P < 0.001). In contrast, eNOS gene transfer significantly reduced RVSP at Day 35 compared with Day 21, whereas VEGF prevented further increases in RVSP over the subsequent 2 wk but did not reverse established PAH. RV hypertrophy was significantly reduced in both the eNOS-treated and VEGF-treated groups compared with the null-transfected controls. Fluorescent microangiography revealed widespread occlusion of the pre-capillary arterioles 21 d after MCT treatment, and animals receiving eNOS gene transfer exhibited the greatest improvement in the arteriolar architecture and capillary perfusion at Day 35. Cell-based eNOS gene transfer was more effective than VEGF in reversing established PAH, associated with evidence of regeneration of pulmonary microcirculation.

Chx10 is required to block photoreceptor differentiation but is dispensable for progenitor proliferation in the postnatal retina.

In the Chx10-null ocular retardation (or(J)) mouse, retinal progenitor cell (RPC) proliferation is impaired, and bipolar neurons, a late born cell type, fail to differentiate. It is unclear whether Chx10 is required to maintain proliferation throughout retinogenesis or whether the bipolar cell defect is an indirect effect of growth arrest. We show that Chx10 is dispensable for late-stage RPC proliferation but is essential to promote bipolar cell genesis in place of rods. Ectopic Chx10 expression drove bipolar instead of rod cell differentiation without affecting division. Converting Chx10 to an activator impaired bipolar cell differentiation, implying that repression is important for Chx10 activity. In the Chx10 null or(J) retina, only a small fraction of cells expressing mutated Chx10 mRNA were rods, but this fraction increased after p27(Kip1) inactivation, which partially rescues proliferation. Most significantly, acute Chx10 knockdown in the postnatal retina promoted rods in place of bipolar neurons without affecting division. Thus, Chx10 directly controls bipolar cell genesis by inhibiting rod differentiation independent of its temporally limited early effect on RPC proliferation.

Fluorescent microangiography (FMA): an improved tool to visualize the pulmonary microvasculature.

Visualization of the complex lung microvasculature and resolution of its three-dimensional architecture remains a difficult experimental challenge. We present a novel fluorescent microscopy technique to visualize both the normal and diseased pulmonary microvasculature. Physiologically relevant pulmonary perfusion conditions were applied using a low-viscosity perfusate infused under continuous airway ventilation. Intensely fluorescent polystyrene microspheres, confined to the vascular space, were imaged through confocal optical sectioning of 200 microm-thick lung sections. We applied this technique to rat lungs and the markedly enhanced depth of field in projected images allowed us to follow vascular branching patterns in both normal lungs and lungs from animals with experimentally induced pulmonary arterial hypertension. In addition, this method allowed complementary immunostaining and identification of cellular components surrounding the blood vessels. Fluorescent microangiography is a widely applicable and quantitative tool for the study of vascular changes in animal models of pulmonary disease.

A novel model for post-transplant obliterative airway disease reveals angiogenesis from the pulmonary circulation.

We present a novel animal model for post-transplant obliterative airway disease in which the donor trachea is implanted into the recipient's lung parenchyma. Although this procedure is technically more challenging than the heterotopic model of implantation into a subcutaneous pouch, it has several important advantages some of which are the appropriate local environment and the possibility of local immunosuppressive therapy after transtracheal gene, cell or drug delivery. This model has revealed new insights into angiogenic potential of the pulmonary circulation.

Differential expression of methyl CpG-binding domain containing factor MBD3 in the developing and adult rat brain.

Using subtractive hybridization screening, the methyl CpG-binding domain containing protein MBD3 was identified as being more prevalently expressed in the embryonic brain than in the adult. In this report, we present the mRNA and protein expression patterns of MBD3 in the developing brain. MBD3 expression was detected in neuroepithelial cells of the developing forebrain, and in peripheral tissues such as liver and intestine during late embryogenesis. This is in contrast to its related family member MBD2, which displayed only minimal expression in the embryonic brain. Immunoblot analysis revealed that the levels of both MBD3 splice forms decrease in the maturing postnatal hippocampus and cortex, although the two forms do not decline at equivalent rates. Immunohistochemical analysis revealed strong MBD3 immunostaining in principal neurons of the hippocampus and cortex, but weak or nondetectable immunostaining in outer cortical layer cells. MBD3 was also selectively expressed in the adult retina, where strong immunoreactivity was detected in cells of the inner nuclear and ganglion cell layers, but no immunoreactivity was detected in cells of the outer nuclear layer. Taken together, these results illustrate that MBD3 displays a selective spatial and temporal pattern of expression in the embryonic and adult brain, thereby strengthening the possibility of MBD3 playing an important role in neuronal development.