Understanding metabolic changes in aging bone marrow.

BACKGROUND: Aging is associated with complex molecular alterations at the cellular level. Bone marrow exhibits distinct phenotypic, genetic and epigenetic alterations with aging. Metabolic changes in the bone marrow related to aging have not been studied. METHODS: In this study, we characterized the metabolome and transcriptome of aging murine bone marrow and compared it with bone marrow from young healthy mice and chemotherapy treated mice; chemotherapy treatment is known to induce age-related changes in hematopoiesis. RESULTS: The metabolome of the aging bone marrow exhibited a signature of suppressed fatty-acid oxidation: accumulation of free fatty acids, reduced acyl-carnitines and low ß-hydroxy butyric acid. The aged bone marrow also exhibited a significant reduction in amino acid and nucleic acid pool. The transcriptome of the aging bone marrow revealed a signature of oxidative stress, known to be associated with mitochondrial dysfunction. Lastly, the metabolic and transcriptomic profiles of the bone marrow of chemotherapy treated mice did not show broad age-related changes but rather mostly resembled young healthy mice, suggestive of a lack of 'metabolic aging' with chemotherapy exposure. CONCLUSION: Our results revealed broad changes in lipids, amino acids, and nucleotides in aging marrow tissue. Together, these data provide a rich resource for the study of metabolic changes associated with aging in bone marrow.

Acute podocyte vascular endothelial growth factor (VEGF-A) knockdown disrupts alphaVbeta3 integrin signaling in the glomerulus.

Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGF(KD)) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ~20% of non-induced controls and urine VEGF-A to ~30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alpha(V)beta(3) integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta(3) integrin and neuropilin-1 in the kidney in vivo and in VEGF(KD) podocytes. Podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF(KD) podocytes downregulates fibronectin and disrupts alpha(V)beta(3) integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha(V)beta(3) integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.

Endothelial nitric oxide synthase, angiotensin-converting enzyme and angiotensinogen gene polymorphisms in hypertensive disorders of pregnancy.

We investigated the variations in genes encoding endothelial nitric oxide synthase (NOS3), angiotensin-converting enzyme (ACE) and angiotensinogen (AGT) in hypertensive disorders of pregnancy and the relationship between the polymorphisms and circulating nitric oxide (NO) and ACE levels in pregnant north Indian women. Frequencies of NOS3 G894T, 4b/a and T(-786) --> C, AGT T704C and ACE ins/del polymorphisms were studied in 342 subjects: 120 with preeclampsia (PE), 104 with gestational hypertension and 118 normotensive pregnant women. Variations were evaluated by polymerase chain reaction-restriction fragment length polymorphism. NO and ACE levels were determined using ELISA. There was no difference in the distribution of individual NOS3 and ACE polymorphisms in the study groups. Haplotype analysis showed a global difference in the NOS3 haplotype distribution between the PE and non-PE subjects (P=0.03). The presence of AGT 704C allele was associated with a reduced risk of developing PE (odds ratio: 0.33, 95% CI: 0.19-0.59 in recessive mode). Circulating total NO and ACE levels were similar in three groups. No relationship was found between circulating NO levels and any of the NOS3 polymorphisms, but the circulating ACE levels were higher in those with DD genotype (P<0.05). In conclusion, there was no association between individual NOS3 and the ACE gene polymorphisms and hypertensive disorders of pregnancy in north Indian women. The presence of minor alleles at all the three sites in NOS3 seemed to increase the risk of PE, and AGT 704C allele was associated with a reduced PE risk. The complexity of interaction between these genetic abnormalities requires further studies.

Maternal EDN1 G5665T polymorphism influences circulating endothelin-1 levels and plays a role in determination of preeclampsia phenotype.

OBJECTIVE: Preeclampsia, a pregnancy-specific syndrome characterized by hypertension and proteinuria, has a familial tendency and is an important cause of maternal and neonatal mortality. Abnormal endothelin-1 synthesis and/or release can explain abnormalities seen in preeclampsia. We prospectively evaluated whether endothelin-1 levels are increased in preeclampsia, to verify if placenta is the source, and any association between the maternal EDN1 G5665T single-nucleotide polymorphism and circulating endothelin-1 levels and preeclampsia manifestation. METHODS: A total of 120 with preeclampsia and 118 normotensive primigravid patients with singleton pregnancy were enrolled. Preeclampsia was defined as new onset of elevated blood pressure greater than 140/90 mmHg and at least 2+ proteinuria on two occasions at least 4 h apart after 20 weeks of gestation in previously normotensive pregnant women. Patients were excluded if they had hypertension before 20 weeks of gestation, diabetes, asthma, heart disease, kidney disease, hematological disorder, autoimmune disease, urinary tract infection, current or past history of smoking, twin pregnancy, molar pregnancy or eclampsia. Genotyping was done using a PCR-RFLP-based method. Placenta samples were collected from 20 preeclampsia and 24 normotensive pregnancies. Reverse transcriptase PCR was done for preproendothelin and beta-actin mRNA. Placental endothelin-1 staining was assessed by immunohistochemistry in villous and extravillous trophoblasts and endothelium. Plasma endothelin-1 levels were measured using ELISA. RESULTS: The preeclampsia group showed higher circulating endothelin-1 levels (1.45 +/- 0.55 vs. 0.91 +/- 0.42 pg/ml; P < 0.0001), reduced frequency of GG genotype (34 vs. 49%; P = 0.025) and increased T allele frequency (0.43 vs. 0.28; P = 0.04). A significant association was noted between endothelin-1 levels and blood pressure in the entire cohort and in the group with preeclampsia (P < 0.001). Circulating endothelin-1 levels were higher in those bearing even one copy of the T variant (1.08 +/- 0.48 vs. 1.31 +/- 0.59 pg/ml; P = 0.004). Placental endothelin-1/beta-actin mRNA ratio was significantly reduced (0.91 +/- 0.77 vs. 3.20 +/- 1.68; P < 0.001) and endothelin-1 staining was lower (P < 0.001) in placental endothelium in preeclampsia. CONCLUSION: Maternal endothelin-1 is elevated and correlates with the severity of blood pressure elevation in preeclampsia. The endothelin-1 is likely released from the maternal endothelium. Presence of T allele at 5665 position in maternal EDN1 gene is associated with higher endothelin-1 levels. Placental endothelin-1 synthesis is reduced in preeclampsia. The combination of elevated maternal and reduced placental endothelin-1 may be an adaptive response to reduced uteroplacental flow in preeclampsia.