Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animal

Vav3 collaborates with p190-BCR-ABL in lymphoid progenitor leukemogenesis, proliferation, and survival

Despite the introduction of tyrosine kinase inhibitor therapy, the prognosis for p190-BCR-ABL(+) acute lymphoblastic leukemia remains poor. In the present study, we present the cellular and molecular roles of the Rho GTPase guanine nucleotide exchange factor Vav in lymphoid leukemogenesis and explore the roles of Vav proteins in BCR-ABL-dependent signaling. We show that genetic deficiency of the guanine nucleotide exchange factor Vav3 delays leukemogenesis by p190-BCR-ABL and phenocopies the effect of Rac2 deficiency, a downstream effector of Vav3. Compensatory up-regulation of expression and activation of Vav3 in Vav1/Vav2-deficient B-cell progenitors increases the transformation ability of p190-BCR-ABL. Vav3 deficiency induces apoptosis of murine and human leukemic lymphoid progenitors, decreases the activation of Rho GTPase family members and p21-activated kinase, and is associated with increased Bad phosphorylation and up-regulation of Bax, Bak, and Bik. Finally, Vav3 activation only partly depends on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to inhibit CrkL activation and impair leukemogenesis in vitro and in vivo. We conclude that Vav3 represents a novel specific molecular leukemic effector for multitarget therapy in p190-BCR-ABL-expressing acute lymphoblastic leukemia

Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type 1 Receptor Expression in db/db Diabetic Mice

Alterations in the renin-angiotensin system (RAS) are considered to be crucial for the development of diabetic complications like hypertension and nephropathy. Our previous work demonstrated role of AT1 receptors (AT1R) in the development of hypertension in db/db diabetic mice. The aim of this study was to test the hypothesis that there is upregulation of renal AT1R and imbalance in renal ACE/ACE2 homeostasis in db/db mice. In addition, we hypothesize that treatment with an anti-hyperglycemic or an AT1R blocker will correct this imbalance. Five week old control and db/db mice were housed in metabolic cages for 24 hour collection of urine. At early age of 5 weeks, db/db mice were obese and hyperglycemic. Urinary albumin excretion was also significantly high in db/db mice. Changes in RAS were evaluated using enzyme activities, western blots and immunohistochemistry. There was a significant increase in urinary ACE2 activity and ACE2 content in db/db mice at 5 weeks. There was a significant increase in plasma ACE activity and Ang II content in db/db mice compared to controls at 8 weeks. Western blot analysis showed significant increase in AT1R protein expression in 8, 18 and 31 week db/db mice compared to controls. There was upregulation of ACE2 and down-regulation of ACE in kidney to compensate the effects of high plasma Ang II. To study the effect of reduction in blood glucose and AT1R blockade, mice were treated with metformin and losartan for 12 weeks. Chronic treatment with metformin (150 mg/kg/day) and losartan (10 mg/kg/day) significantly decreased urinary albumin and protein excretion. Metformin improved blood glucose and glucose tolerance db/db mice, but did not affect renal expression of ACE, ACE2 and AT1R. Although chronic losartan treatment did not alter blood glucose levels, it improved the morphology of pancreatic islets. There was a significant increase in renal AT1R protein expression and decrease in renal ACE2 protein expression following losartan treatment. Losartan treatment significantly increased urinary ACE2 activity. Western blot of concentrated urine from 8 week db/db mice revealed immunoreactive bands of ACE, ACE2 and AT1R protein. Conclusion: 1) There is upregulation in renal AT1R protein expression in db/db mice. 2) Chronic metformin treatment significantly reduces blood glucose and microalbuminuria in db/db mice without affecting ACE/ACE2 balance. 3) Chronic losartan treatment had no effect on blood glucose, but it up-regulates renal AT1R and down-regulates renal ACE2. 4) Enzyme activity and western blot shows increased excretion of ACE2 in the urine of db/db mice. These data show that urinary ACE and ACE2 provide good index of intra-renal RAS status and could be used in early diagnosis and prognosis of diabetic renal disease

Rosiglitazone Treatment of Type 2 Diabetic db/db Mice Attenuates Urinary Albumin and Angiotensin Converting Enzyme 2 Excretion

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication

Rosiglitazone Treatment of Type 2 Diabetic db/db Mice Attenuates Urinary Albumin and Angiotensin Converting Enzyme 2 Excretion

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication

Immunofluorescence of nephrin, ACE2 and ADAM17 after 8 weeks of treatment with rosiglitazone.

<p>(A) Immunofluorescence staining for nephrin in the glomeruli of control, untreated and rosiglitazone treated <i>db/db</i> mice at 20× magnification. Nephrin expression was significantly decreased in <i>db/db</i> mice. After eight weeks of treatment with rosiglitazone there was a significant increase in nephrin expression compared to untreated <i>db/db</i> mice. *<i>p</i><0.01 Vs control mice.^#<i>p</i><0.05 Vs untreated <i>db/db</i> mice. Each bar represents mean ± SEM of group size (n = 11–18). (B) Immunofluorescence staining for ACE2 in cortical tubules and glomeruli of control, untreated and rosiglitazone treated <i>db/db</i> mice at 20× magnification. White arrows indicate glomeruli. While tubular ACE2 expression was increased, glomerular ACE2 expression was significantly decreased in <i>db/db</i> mice. After eight weeks of treatment with rosiglitazone there was a significant increase in glomerular ACE2 expression while tubular ACE2 expression was unchanged compared to untreated <i>db/db</i> mice. *<i>p</i><0.001 Vs control mice.^#<i>p</i><0.01 Vs untreated <i>db/db</i> mice. Each bar represents mean ± SEM of group size (n = 11–18). (C) Immunofluorescence staining for ADAM17 in cortical tubules of control, untreated and rosiglitazone treated <i>db/db</i> mice at 20× magnification. (D) Immunofluorescence double staining for ACE2 and ADAM17 in cortical tubules of <i>db/db</i> mice at 60× magnification.</p

ACE2 and ACE activity in urine, plasma and kidney of control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice using a fluorometric enzyme assay.

<p>(A) Urinary ACE2 activity in control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice before and after the commencement of treatment. Two-way ANOVA showed an increase in urinary ACE2 activity of <i>db/db</i> mice compared to control mice. Four and eight weeks after treatment commenced there was a significant decrease in urinary ACE2 activity of the <i>db/db</i>+rosiglitazone mice compared to untreated <i>db/db</i> mice. *<i>p</i><0.001 Vs control mice.^#<i>p</i><0.001 Vs untreated <i>db/db</i> mice. Each bar represents mean ± SEM of group size (n = 6–7). (B) Plasma and renal ACE2 activity in control, <i>db/db</i> and <i>db/db+</i>rosiglitazone mice. There was no plasma ACE2 activity in control and <i>db/db</i> mice but a significant increase in renal ACE2 activity of <i>db/db</i> mice compared to control mice was observed. Treatment with rosiglitazone had no significant effect on renal ACE2 activity of treated <i>db/db</i> mice compared to untreated <i>db/db</i> mice.*<i>p</i><0.05 Vs control kidney. Each bar represents mean ± SEM of group size (n = 5–8). (C) Plasma ACE activity in control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice 8 wks after the commencement of treatment. One-way ANOVA showed an increase in plasma ACE activity of <i>db/db</i> mice compared to control mice. Eight weeks after treatment commenced there was a significant decrease in plasma ACE activity of the <i>db/db</i>+rosiglitazone mice compared to untreated <i>db/db</i> mice. *<i>p</i><0.05, **<i>p</i><0.001 Vs control mice.^#<i>p</i><0.05 Vs untreated <i>db/db</i> mice. Each bar represents mean ± SEM of group size (n = 6–7).</p

Mass spectrometric analysis of ACE2 activity in urine from control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice.

<p>Urine (2 µl) was incubated for 1.5 h at 37°C in 50 mM MES buffer pH 6.75 containing 0.5 µM Ang II, 2 mM PMSF and 20 µM bestatin. Shown is the conversion of Ang II (<i>m/z</i> 1046) to Ang-(1–7) (<i>m/z</i> 899). (A) Urinary ACE2 activity in control mice. (B) Urinary ACE2 activity in <i>db/db</i> mice. (C) Urinary ACE2 activity in <i>db/db</i> mice treated with rosiglitazone. (D) Urinary ACE2 activity in <i>db/db</i> mice in incubations with the ACE2 inhibitor, MLN-4760. (E) MS/MS of enzymatically generated Ang-(1–7) (upper panel) and synthetic Ang-(1–7) (lower panel).</p

Linear regression analysis between urinary ACE2 activity and urinary albumin, plasma glucagon, plasma triglycerides and blood glucose levels.

<p>(A) Association of urinary ACE2 activity and urinary albumin excretion in control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice before and 4 or 8 weeks after commencement of rosiglitazone treatment. (B) Correlation between plasma glucagon and urinary ACE2 activity in control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice 8 weeks after commencement of rosiglitazone treatment. (C) Correlation between plasma triglycerides and urinary ACE2 activity in control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice 8 weeks after commencement of rosiglitazone treatment. (D) Correlation between urinary ACE2 activity and non fasted blood glucose levels in control, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice 8 weeks after commencement of rosiglitazone treatment.</p

Age dependent changes in general metabolic parameters of control, control+rosiglitazone, <i>db/db</i> and <i>db/db</i>+rosiglitazone mice.

<p>Values represent mean ± SEM. *<i>p</i><0.05 Vs age-matched control mice, ^$<i>p</i><0.05 Vs. age-matched control+rosiglitazone mice and ^#<i>p</i><0.05 Vs age-matched <i>db/db</i> mice were considered statistically significant. ND means not determined. ^a The amount of food spilled was minimal and was not accounted for in the data presented in the table.</p