We reported recently that chronic thyroid deficiency in rat, beginning in utero and terminating after maturity, suppresses lesion-induced central catecholaminergic axon sprouting in the adult brain [Gottesfeld et al, 1985]. The present work was undertaken to define the critical period of hypothyroidism on subsequent neuronal sprouting. Thyroid hormones deficiency was induced in rats by methimazole during (a) gestational days 8-21 (20 mg/kg/day in the drinking water); (b) postnatal days 1-15 (0.2 or 0.4 mg/pup/day; i.p.), or (c) in the mature animal for 4 weeks (20 mg/kg/day in the drinking water). The olfactory tubercles (OTs) were used as a model to study sprouting of dopaminergic (DA) nerve terminals, elicited by olfactory bulbectomy. Animals in each group received lesions or sham operations as adults, and sacrificed 3 weeks after the operation. Thus, for each of the above treatments four subgroups were formed: (a) euthyroid/sham-operation, (b) euthyroid/lesion, (c) hypothyroid/sham-operation, and (d) hypothyroid/lesion. Sprouting of DA axon terminals in the OTs was identified by biochemical assays and quantitative immunofluorescent microscopy, using tyrosine hydroxylase (TH) as a marker. Serum thyroxine levels served as an index of the thyroid status. The results demonstrate that lesion-induced sprouting of DA axon terminals in OTs of adult rats is suppressed by hypothyroidism induced prenatally or during the early postnatal period, but not after maturity. Thus, there is a perinatal critical period during which altered thyroid function exerts long-term effects on neuronal plasticity.
Bone marrow‐derived hematopoietic stem progenitor cells (HSPCs) have vasoreparative functions and are physiologically mobilized into circulation. Aging or diabetes increases risk for cardiovascular diseases and are known to be associated with impaired HSPC functions. Angiotensin converting enzyme (ACE) and ACE2 are primary enzymes of cardiovascular detrimental and protective axes of renin‐angiotensin system (RAS). Angiotensin‐(1‐7) is generated largely by degradation of Ang II by ACE2 and produces vasoprotective functions by activating Mas receptor. We have shown that Ang‐(1‐7) reverses diabetic dysfunction in murine and human HSPCs. This study tested the hypothesis that aging healthy or with diabetes is associated with imbalance in the vascular protective and detrimental axes of RAS in HSPCs. Peripheral blood samples were obtained from either male or female healthy (n=23) or diabetic (n=21), both type 1 and type 2, individuals of age ranging from 25 to 87 years. Lineage‐negative (Lin−) or CD34+ HSPCs were enriched from mononuclear cells. ACE and ACE2 activities in the cell lysates were determined by using enzyme‐specific fluorogenic substrates. Enzyme activities are expressed as arbitrary fluorescence units that is inhibitable by the enzyme‐selective inhibitors of ACE or ACE2, captopril and MLN‐4760, respectively. ACE2/ACE activity ratios in HSPCs in different individuals are plotted against the respective ages. ACE2/ACE is negatively correlated with the age in healthy (Pearson's r=−0.88, P<0.0001) and in diabetic individuals (Pearson's r=−0.82, P<0.0001). As predicted, a negative correlation was observed with ACE2/ACE vs HbA1C (Pearson's r=−0.899, P<0.0001). These results imply that vascular protective axis of RAS is dysfunctional in HSPCs of aging individuals either healthy or diabetic, which is likely to be an underlying mechanism of the impaired reparative functions in HSPCs. Molecular or pharmacological approaches that enhance ACE2 activity or expression could be of great promise in reversing the age‐ or diabetes‐associated dysfunctions and restore cardiovascular health.Support or Funding InformationNational Institute of General Medical Sciences (NIGMS), and National Institute of Aging (AG056881)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hematopoietic stem/progenitor cells (HS/PCs) have the propensity of ischemic vascular repair, and this innate vasoprotective function is impaired in diabetes. Angiotensin-converting enzyme (ACE) and ACE-2 are primary enzymes of the cardiovascular-detrimental and protective axes of the renin-angiotensin system, respectively. In this study, we tested the hypothesis that diabetic dysfunction in HS/PCs is due to ACE2/ACE-imbalance, and that increasing ACE2 expression will restore the vasoreparative potential. Lineage- (Lin - ) or CD34 + cells were isolated from the peripheral blood mononuclear cells (MNCs) of nondiabetic subjects (ND), and type 1 or type 2 diabetic (DB) patients (male or female, age 48-76 years, HbA1C 6.5-11.2). ACE and ACE2 activities were measured in lysates of MNCs, Lin - and CD34 + cells by enzyme-selective fluorogenic substrates and inhibitors. Lentiviral ACE2 (LV-ACE2) gene transfer was carried out by spinoculation. Reparative function of Lin - cells was evaluated in Foxn1 nu mice undergoing hind limb ischemia (HLI). Lin - cells with or without ACE2-overexpression were administered (i.m) in the peri-ischemic region, and the blood flow recovery was monitored. Circulating Lin - or CD34 + cells are lower in DB (Lin - ((2±0.2)х10 5 , P<0.04) and CD34 + ((0.6±0.1)х10 3 , P<0.02), n=8) compared to ND group (Lin - (25±4)х10 5 ; CD34 + (2±0.4)х10 3 per 10 6 MNCs, n=8). This was associated with decrease in ACE2 and increase in ACE activity, resulting in 4.5-fold decrease in ACE2/ACE ratio in DB-CD34 + cells (0.4±0.07 vs ND 1.8±0.2, P<0.01, n=6). This was negatively correlated with HbA1C (r 2 =0.89, P<0.01). Administration of ND-Lin - cells has no effect on the blood flow recovery of mice following HLI. In contrast, administration of DB-Lin - cells decreased this recovery (48±6%, n=5, P<0.001 vs ND-Lin - 105±7%). However, treatment with ACE2-overexpressing DB-Lin - cells robustly enhanced blood flow recovery (112±8%, n=4, P<0.001 vs DB). These observations suggest that ACE2/ACE imbalance is correlated with diabetic vasoreparative dysfunction in HS/PCs, and that increasing ACE2 expression reverses the dysfunction. ACE2 gene transfer is a promising approach for enhancing vascularization outcomes of cell-based therapies in diabetic individuals.
Diabetic retinopathy (DR) is a common cause of vision loss and blindness. Healthy CD34+ stem cells are capable of homing to vascular lesions and facilitating vascular repair. However, many diabetic patients have dysfunctional CD34+ stem cells with no reparative potential. CD34+ dysfunction is corrected by transiently inhibiting endogenous transforming growth factor-β1 (TGF-β1) within the patient's own dysfunctional CD34+ stem cells using phosphorodiamidate morpholino oligomers (PMOs). Antisense TGF-β1treated dysfunctional CD34+ stem cells are now functional, no longer require growth factor stimulation to evade apoptosis, and are stable at 37 C ex vivo for >5 days. We identified three markers of restored stem cell function: (1) upregulation of CXCR4 expression necessary for stem cell homing and adhesion, (2) SDF-1-mediated nitric oxide (NO) production required for cell mobility, and (3) restoration of the ability of CD34+ cells to migrate and repair vascular lesions. The antisense targets autocrine TGF-β expression, whereas neutralizing antibodies do not. The PMO antisense triggers a cascade of hematopoietic proliferation and differentiation that paracrine TGF-β cannot alter. We describe optimal PMO manipulation of CD34+ stem cells ex vivo for transplantation, screening multiple gene targets leading to the identification of TGF-β1, and a lead TGF-β1 inhibitor evaluated in clinical studies.
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