Endothelial precursor cells (EPCs

The present epidemic of diabetes is resulting in a worldwide increase in cardiovascular and microvascular complications including retinopathy. Current thinking has focused on local influences in the retina as being responsible for development of this diabetic complication. However, the contribution of circulating cells in maintenance, repair, and dysfunction of the vasculature is now becoming appreciated. Diabetic individuals have fewer endothelial progenitor cells (EPCs) in their circulation and these cells have diminished migratory potential, which contributes to their decreased reparative capacity. Using a rat model of type 2 diabetes, we show that the decrease in EPC release from diabetic bone marrow is caused by bone marrow neuropathy and that these changes precede the development of diabetic retinopathy. In rats that had diabetes for 4 mo, we observed a dramatic reduction in the number of nerve terminal endings in the bone marrow. Denervation was accompanied by increased numbers of EPCs within the bone marrow but decreased numbers in circulation. Furthermore, denervation was accompanied by a loss of circadian release of EPCs and a marked reduction in clock gene expression in the retina and in EPCs themselves. This reduction in the circadian peak of EPC release led to diminished reparative capacity, resulting in the development of the hallmark feature of diabetic retinopathy, acellular retinal capillaries. Thus, for the first time, diabetic retinopathy is related to neuropathy of the bone marrow. This novel finding shows that bone marrow denervation represents a new therapeutic target for treatment of diabetic vascular complications.

Rationale : Studies have demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a protective role against lung diseases, including pulmonary hypertension (PH). Recently, an antitrypanosomal drug, diminazene aceturate (DIZE), was shown to exert an "offtarget" effect of enhancing the enzymatic activity of ACE2 in vitro. Objectives: To evaluate the pharmacological actions of DIZE in experimental models of PH. Methods: PH was induced in male Sprague Dawley rats by monocrotaline, hypoxia, or bleomycin challenge. Subsets of animals were simultaneously treated with DIZE. In a separate set of experiments, DIZE was administered after 3 weeks of PH induction to determine whether the drug could reverse PH. Measurements and Main Results: DIZE treatment significantly prevented the development of PH in all of the animal models studied. The protective effects were associated with an increase in the vasoprotective axis of the lung renin-angiotensin system, decreased inflammatory cytokines, improved pulmonary vasoreactivity, and enhanced cardiac function. These beneficial effects were abolished by C-16, an ACE2 inhibitor. Initiation of DIZE treatment after the induction of PH arrested disease progression. Endothelial dysfunction represents a hallmark of PH pathophysiology, and growing evidence suggests that bone marrow-derived angiogenic progenitor cells contribute to endothelial homeostasis. We observed that angiogenic progenitor cells derived from the bone marrow of monocrotaline-challenged rats were dysfunctional and were repaired by DIZE treatment. Likewise, angiogenic progenitor cells isolated from patients with PH exhibited diminished migratory capacity toward the key chemoattractant stromal-derived factor 1a, which was corrected by in vitro DIZE treatment. Conclusions: Our results identify a therapeutic potential of DIZE in PH therapy.Keywords: pulmonary hypertension; ACE2; angiogenic progenitor cells; diminazene Pulmonary hypertension (PH) is a life-threatening disease characterized by elevated pressure in the pulmonary arteries and What This Study Adds to the FieldWe show that diminazene, an antitrypanosomal drug, attenuates hemodynamic changes, prevents maladaptive right ventricular remodeling, and enhances pulmonary vasorelaxation in experimental models of PH through activation of ACE2. Furthermore, diminazene improves the functions of APCs obtained from experimental animals and patients with PH. This study identifies a new application for an existing drug, which could be successfully developed for PH therapeutics.

In the present work, we reported a new nuclear Overhauser enhancement (NOE)-mediated magnetization transfer (MT) signal at around −1.6 ppm (NOE(−1.6)) in rat brain and investigated its application in the detection of acute ischemic stroke in rodent model. Using continuous wave (CW) MT sequence, the NOE(−1.6) is reliably detected in rat brain. The amplitude of this new NOE signal in rat brain was quantified using a 5-pool Lorentzian Z-spectral fitting method. Amplitudes of amide, amine, NOE at −3.5 ppm (NOE(−3.5)), as well as NOE(−1.6) were mapped using this fitting method in rat brain. Several other conventional imaging parameters (R 1 , R 2 , apparent diffusion coefficient (ADC), and semi-solid pool size ratio (PSR)) were also measured. Our results showed that NOE(−1.6), R 1 , R 2 , ADC, and APT signals from stroke lesion have significant changes at 0.5-1 h after stroke. Compared with several other imaging parameters, NOE(−1.6) shows the strongest contrast differences between stroke and contralateral normal tissues and stays consistent over time until 2 h after onset of stroke. Our results demonstrate that this new NOE(−1.6) signal in rat brain is a new potential contrast for assessment of acute stroke in vivo and might provide broad applications in the detection of other abnormal tissues.

Autonomic nervous system (ANS) dysfunction, exaggerated inflammation and impaired vascular repair are all hallmarks of hypertension. Considering the bone marrow (BM) is a major source of the inflammatory cells (ICs) and endothelial progenitor cells (EPCs), we hypothesized that impaired BM-ANS interaction contributes to dysfunctional BM activity in hypertension. In the SHR, we observed a >30% increase in BM and blood ICs (CD4.8+), and a >50% decrease in EPCs (CD90+.CD4.5.8-) compared to the normotensive Wistar-Kyoto (WKY) rat. Increased tyrosine hydroxylase (70%) and norepinephrine (NE, 160%), and decreased choline acetyl transferase (30%) and acetylcholine esterase (55%) indicated imbalanced ANS in SHR BM. In WKY, night time-associated elevation in SNA (50%) and BM NE (41%) was associated with increased ICs (50%) and decreased EPCs (350%), while BM sympathetic denervation decreased ICs (25%) and increased EPCs (40%). In contrast, these effects were blunted in SHR, possibly due to chronic downregulation of BM adrenergic receptor α2a (by 50-80%) and β2 (30-45%). Application of NE resulted in increased BM IC activation/release, which was prevented by pre-administration of Ach. Electrophysiological recordings of femoral SNA (fSNA) showed a more robust fSNA activity in SHR compared to WKY, peaking earlier in the respiratory cycle, indicative of increased sympathetic tone. Finally, manganese-enhanced magnetic resonance imaging (MEMRI) demonstrated that pre-sympathetic neuronal activation in SHR was associated with an accelerated retrograde transport of the GFP-labeled pseudorabies virus from the BM. These observations demonstrate that a dysfunctional BM ANS is associated with imbalanced EPCs and ICs in hypertension.

Previously we demonstrated that central administration of angiotensin-(1-7) [Ang-(1-7)] into rats elicits significant cerebroprotection against ischemic stroke elicited by endothelin-1 induced middle cerebral artery occlusion. Ang-(1-7), acting via its receptor Mas, reduced cerebral infarct size, and rats exhibited improved performance on neurological exams. These beneficial actions of Ang-(1-7) were not due to inhibition of the effects of endothelin-1 on cerebral vasoconstriction or effects on cerebral blood flow, and so we considered other potential mechanisms. Here we investigated the possibility that the Ang-(1-7)-induced cerebroprotection involves an anti-inflammatory effect, since stroke-induced cerebral damage includes an excessive intracerebral inflammatory response. Our quantitative RT-PCR analyses revealed that central Ang-(1-7) treatment attenuates the increased expression of mRNAs for inducible nitric oxide synthase (iNOS), several pro-inflammatory cytokines and cluster of differentiation molecule 11b (microglial marker) within the cerebral cortex following endothelin-1 induced stroke. Western blotting confirmed similar changes in iNOS protein expression in the cerebral cortex. In support of these observations, immunostaining revealed the presence of immunoreactive Mas on activated microglia within the cerebral cortical infarct zone, and in vitro experiments demonstrated that lipopolysaccharide-induced increases in nitric oxide production in glial cultures are attenuated by Ang-(1-7) acting via Mas. Collectively these findings demonstrate an anti-inflammatory action of Ang-(1-7) in the brain, and suggest that the cerebroprotective action of this peptide in ischemic stroke may involve effects on nitric oxide generation by microglia.

We asked whether the hypoxia-regulated factor, insulin-like growth factor binding protein-3 (IGFBP3), could modulate stem cell factor receptor (c-kit ؉ ), stem cell antigen-1 (sca-1 ؉ ), hematopoietic stem cell (HSC), or CD34 ؉ endothelial precursor cell (EPC) function. Exposure of CD34 ؉ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygeninduced retinopathy (OIR) model. In separate studies, GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration, protection of the developing vasculature from hyperoxia-induced regression, and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion, IGFBP3 mediates EPC migration, differentiation, and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions. IGFBP3 ͉ angiogenesis ͉ retinopathy of prematurity V ascular damage associated with diabetic retinopathy and retinopathy of prematurity (ROP) results from tissue ischemia, and, subsequently, this ischemia leads to development of pathological neovascularization. Insulin-like growth factor 1 (IGF1) is required for normal retinal vascular development because vascular development is arrested in its absence despite the presence of VEGF (1). Development of ROP is associated with low levels of IGF1 (2) because the lack of IGF1 in the early neonatal period leads to the development of avascular retina, which results in ROP (3). However, unregulated IGF1 expression can lead to pathological neovascularization (4-13), and IGF1 receptor (IGF1R) antagonists are able to suppress retinal neovascularization in vivo by inhibiting VEGF signaling (1).The effects of IGF1 are mediated by IGF1R and modulated by complex interactions with IGF binding proteins (IGFBPs), which are also modulated at multiple levels. Six IGFBPs function as transporter proteins and storage pools for IGF1 in a tissue-and developmental stage-specific manner. Phosphorylation, proteolysis, polymerization (8), and cell or matrix association (9) regulates the functions of IGFBPs. Specific IGFBPs have been shown to either stimulate or inhibit IGF1 action (10).IGFBP3, the best studied and most abundant of these binding proteins, carries Ն75% of serum IGF1 and IGF2 in heterotrimeric complexes. Besides its endocrine effects, IGFBP3 has auto-and paracrine actions affecting cell mobility, adhesion, apoptosis, survival, and the cell cycle (14,15). Like the other IGFBPs, IGFBP3 has IGF1-in...

Diabetic retinopathy is the leading cause of blindness in working-age adults. It is caused by oxygen starvation in the retina inducing aberrant formation of blood vessels that destroy retinal architecture. In humans, vitreal stromal cell-derived factor-1 (SDF-1) concentration increases as proliferative diabetic retinopathy progresses. Treatment of patients with triamcinolone decreases SDF-1 levels in the vitreous, with marked disease improvement. SDF-1 induces human retinal endothelial cells to increase expression of VCAM-1, a receptor for very late antigen-4 found on many hematopoietic progenitors, and reduce tight cellular junctions by reducing occludin expression. Both changes would serve to recruit hematopoietic and endothelial progenitor cells along an SDF-1 gradient. We have shown, using a murine model of proliferative adult retinopathy, that the majority of new vessels formed in response to oxygen starvation originate from hematopoietic stem cell-derived endothelial progenitor cells. We now show that the levels of SDF-1 found in patients with proliferative retinopathy induce retinopathy in our murine model. Intravitreal injection of blocking antibodies to SDF-1 prevented retinal neovascularization in our murine model, even in the presence of exogenous VEGF.Together, these data demonstrate that SDF-1 plays a major role in proliferative retinopathy and may be an ideal target for the prevention of proliferative retinopathy.