Spatiotemporal heterogeneity and patterning of developing renal blood vessels.

The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of specification by E13.5 when the first arteries can be identified. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immunofluorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.

Effects of Increased Arterial Stiffness on Atherosclerotic Plaque Amounts.

Increased arterial stiffness is associated with atherosclerosis in humans, but there have been limited animal studies investigating the relationship between these factors. We bred elastin wildtype (Eln+/+) and heterozygous (Eln+/-) mice to apolipoprotein E wildtype (Apoe+/+) and knockout (Apoe-/-) mice and fed them normal diet (ND) or Western diet (WD) for 12 weeks. Eln+/- mice have increased arterial stiffness. Apoe-/- mice develop atherosclerosis on ND that is accelerated by WD. It has been reported that Apoe-/- mice have increased arterial stiffness and that the increased stiffness may play a role in atherosclerotic plaque progression. We found that Eln+/+Apoe-/- arterial stiffness is similar to Eln+/+Apoe+/+ mice at physiologic pressures, suggesting that changes in stiffness do not play a role in atherosclerotic plaque progression in Apoe-/- mice. We found that Eln+/-Apoe-/- mice have increased structural arterial stiffness compared to Eln+/+Apoe-/- mice, but they only have increased amounts of ascending aortic plaque on ND, not WD. The results suggest a change in atherosclerosis progression but not end stage disease in Eln+/-Apoe-/- mice due to increased arterial stiffness. Possible contributing factors include increased blood pressure and changes in circulating levels of interleukin-6 (IL6) and transforming growth factor beta 1 (TGF-ß1) that are also associated with Eln+/- genotype.

Marrow Adipose Tissue Expansion Coincides with Insulin Resistance in MAGP1-Deficient Mice.

Marrow adipose tissue (MAT) is an endocrine organ with the potential to influence skeletal remodeling and hematopoiesis. Pathologic MAT expansion has been studied in the context of severe metabolic challenge, including caloric restriction, high fat diet feeding, and leptin deficiency. However, the rapid change in peripheral fat and glucose metabolism associated with these models impedes our ability to examine which metabolic parameters precede or coincide with MAT expansion. Microfibril-associated glycoprotein-1 (MAGP1) is a matricellular protein that influences cellular processes by tethering signaling molecules to extracellular matrix structures. MAGP1-deficient (Mfap2 (-/-)) mice display a progressive excess adiposity phenotype, which precedes insulin resistance and occurs without changes in caloric intake or ambulation. Mfap2 (-/-) mice were, therefore, used as a model to associate parameters of metabolic disease, bone remodeling, and hematopoiesis with MAT expansion. Marrow adiposity was normal in Mfap2 (-/-) mice until 6 months of age; however, by 10 months, marrow fat volume had increased fivefold relative to wild-type control at the same age. Increased gonadal fat pad mass and hyperglycemia were detectable in Mfap2 (-/-) mice by 2 months, but peaked by 6 months. The development of insulin resistance coincided with MAT expansion. Longitudinal characterization of bone mass demonstrated a disconnection in MAT volume and bone volume. Specifically, Mfap2 (-/-) mice had reduced trabecular bone volume by 2 months, but this phenotype did not progress with age or MAT expansion. Interestingly, MAT expansion in the 10-month-old Mfap2 (-/-) mice was associated with modest alterations in basal hematopoiesis, including a shift from granulopoiesis to B lymphopoiesis. Together, these findings indicate MAT expansion is coincident with insulin resistance, but not excess peripheral adiposity or hyperglycemia in Mfap2 (-/-) mice; and substantial MAT accumulation does not necessitate a proportional decrease in either bone mass or bone marrow cellularity.

Hypertension and decreased aortic compliance due to reduced elastin amounts do not increase atherosclerotic plaque accumulation in Ldlr-/- mice.

BACKGROUND AND AIMS: High blood pressure and reduced aortic compliance are associated with increased atherosclerotic plaque accumulation in humans. Animal studies support these associations, but additional factors, such as fragmented elastic fibers, are present in most previous animal studies. Elastin heterozygous (Eln+/-) mice have high blood pressure and reduced aortic compliance, with no evidence of elastic fiber fragmentation and represent an appropriate model to directly investigate the effects of these factors on atherosclerosis. METHODS AND RESULTS: Eln+/- and Eln+/+ mice were crossed with low density lipoprotein receptor knockout (Ldlr-/-) and wild-type (Ldlr+/+) mice and fed normal or Western diet (WD) for 16 weeks. We hypothesized that on WD, Eln+/-Ldlr-/- mice with high blood pressure and reduced aortic compliance would have increased atherosclerotic plaque accumulation compared to Eln+/+Ldlr-/- mice. We measured serum cholesterol and cytokine levels, blood pressure, aortic compliance, and plaque accumulation. Contrary to our hypothesis, we found that on WD, Eln+/-Ldlr-/- mice do not have increased plaque accumulation compared to Eln+/+Ldlr-/- mice. At the aortic root, there are no significant differences in plaque area between Eln+/-Ldlr-/- and Eln+/+Ldlr-/- mice on WD (p = 0.89), while in the ascending aorta, Eln+/-Ldlr-/- mice on WD have 29% less normalized plaque area than Eln+/+Ldlr-/- mice on WD (p = 0.009). CONCLUSION: Using an atherogenic mouse model, we conclude that increased blood pressure and reduced aortic compliance are not direct causes of increased aortic plaque accumulation. We propose that additional insults, such as fragmentation of elastic fibers, are necessary to alter plaque accumulation.

Characterization of metabolic health in mouse models of fibrillin-1 perturbation.

Mutations in the microfibrillar protein fibrillin-1 or the absence of its binding partner microfibril-associated glycoprotein (MAGP1) lead to increased TGFß signaling due to an inability to sequester latent or active forms of TGFß, respectively. Mouse models of excess TGFß signaling display increased adiposity and predisposition to type-2 diabetes. It is therefore interesting that individuals with Marfan syndrome, a disease in which fibrillin-1 mutation leads to aberrant TGFß signaling, typically present with extreme fat hypoplasia. The goal of this project was to characterize multiple fibrillin-1 mutant mouse strains to understand how fibrillin-1 contributes to metabolic health. The results of this study demonstrate that fibrillin-1 contributes little to lipid storage and metabolic homeostasis, which is in contrast to the obesity and metabolic changes associated with MAGP1 deficiency. MAGP1 but not fibrillin-1 mutant mice had elevated TGFß signaling in their adipose tissue, which is consistent with the difference in obesity phenotypes. However, fibrillin-1 mutant strains and MAGP1-deficient mice all exhibit increased bone length and reduced bone mineralization which are characteristic of Marfan syndrome. Our findings suggest that Marfan-associated adipocyte hypoplasia is likely not due to microfibril-associated changes in adipose tissue, and provide evidence that MAGP1 may function independently of fibrillin in some tissues.

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Elastin Insufficiency Predisposes Mice to Impaired Glucose Metabolism.

Williams-Beuren syndrome is the consequence of a large contiguous-gene deletion on the seventh human chromosome that includes the elastin gene. Elastin is an extracellular matrix protein responsible for the cardiovascular abnormalities associated with Williams's syndrome, including hypertension and aortic stenosis. A high percentage of individuals with Williams's syndrome also have impaired glucose tolerance, independent of traditional risk factors for diabetes. Here, we show that murine adipose tissue does assemble elastic fibers; however, isolated elastin insufficiency (Eln+/-) in mice does not independently influence glucose metabolism or tissue lipid accumulation. Similarly, isolated ApoE deficiency (ApoE-/-), a model of hyperlipidemia and atherosclerosis, does not impair insulin sensitivity. However, Eln+/-; ApoE-/- double mutant mice exhibit notable hyperglycemia, adipocyte hypertrophy, inflammation of adipose tissue, and ectopic lipid accumulation in liver tissue. Further, Eln+/-; ApoE-/- mutants have significant impairment of insulin sensitivity by insulin tolerance testing, independent of body weight or diet, suggesting that elastin insufficiency predisposes to metabolic disease in susceptible individuals.