Differential extracellular vesicle concentration and their biomarker expression of integrin αv/ß5, EpCAM, and glypican-1 in pancreatic cancer models.

Tumor-derived extracellular vesicles (EVs) show great potential as biomarkers for several diseases, including pancreatic cancer, due to their roles in cancer development and progression. However, the challenge of utilizing EVs as biomarkers lies in their inherent heterogeneity in terms of size and concentration, making accurate quantification difficult, which is highly dependent on the isolation and quantification methods used. In our study, we compared three EV isolation techniques and two EV quantification methods. We observed variations in EV concentration, with approximately 1.5-fold differences depending on the quantification method used. Interestingly, all EV isolation techniques consistently yielded similar EV quantities, overall size distribution, and modal sizes. In contrast, we found a notable increase in total EV amounts in samples from pancreatic cancer cell lines, mouse models, and patient plasma, compared to non-cancerous conditions. Moreover, individual tumor-derived EVs exhibited at least a 3-fold increase in several EV biomarkers. Our data, obtained from EVs isolated using various techniques and quantified through different methods, as well as originating from various pancreatic cancer models, suggests that EV profiling holds promise for the identification of unique and cancer-specific biomarkers in pancreatic cancer.

The role of telomerase reverse transcriptase in the mitochondrial protective functions of Angiotensin-(1-7) in diabetic CD34+ cells.

Angiotensin (Ang)-(1-7) stimulates vasoprotective functions of diabetic (DB) CD34+ hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS), increasing nitric oxide (NO) levels and decreasing TGFß1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-, ß- and α-ß-TERT, which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1-7) or TGFß1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing. CD34+ cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND, n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT). TERT inhibitor or mitoXTERT prevented the effects of Ang-(1-7) on NO or mitoROS levels in DB-CD34+ cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1-7) or TGFß1-silencing. The prevalence of TERT splice variants, with predominant ß-TERT expression, was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1-7) or TGFß1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of ß-splicing increased FL-TERT and protected mitoDNA in DB-cells. The findings suggest that diabetes induces TERT-splicing in CD34+ cells and that ß-TERT splice variant largely contributes to the mitoDNA oxidative damage.

Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1-7).

Compromised barrier function of colon epithelium with aging is largely due to gut microbial dysbiosis. Recent studies implicate an important role for angiotensin converting enzymes, ACE and ACE2, angiotensins, and the receptors, AT1 receptor (AT1R) and Mas receptor (MasR), in the regulation of colon functions. The present study tested the hypothesis that leaky gut in aging is associated with an imbalance in ACE2/ACE and that the treatment with angiotenisn-(1-7) (Ang-(1-7)) will restore gut barrier integrity and microbiome. Studies were carried out in Young (3-4 months) and old (20-24 months) male mice. Ang-(1-7) was administered by using osmotic pumps. Outcome measures included expressions of ACE, ACE2, AT1R, and MasR, intestinal permeability by using FITC-dextran, and immunohistochemistry of claudin 1 and occludin, and intestinal stem cells (ISCs). ACE2 protein and activity were decreased in Old group while that of ACE were unchanged. Increased intestinal permeability and plasma levels of zonulin-1 in the Old group were normalized by Ang-(1-7). Epithelial disintegrity, reduced number of goblet cells and ISCs in the old group were restored by Ang-(1-7). Expression of claudin 1 and occludin in the aging colon was increased by Ang-(1-7). Infiltration of CD11b+ or F4/80+ inflammatory cells in the old colons were decreased by Ang-(1-7). Gut microbial dysbiosis in aging was evident by decreased richness and altered beta diversity that were reversed by Ang-(1-7) with increased abundance of Lactobacillus or Lachnospiraceae. The present study shows that Ang-(1-7) restores gut barrier integrity and reduces inflammation in the aging colon by restoring the layer of ISCs and by restructuring the gut microbiome.

The Effect of Direct and Remote Postexercise Ischemic Conditioning on Muscle Soreness and Strength 24 Hours After Eccentric Drop Jumps.

ABSTRACT: Lillquist, T, Mahoney, SJ, Kotarsky, C, McGrath, R, Jarajapu, Y, Scholten, SD, and Hackney, KJ. The effect of direct and remote postexercise ischemic conditioning on muscle soreness and strength 24 hours after eccentric drop jumps. J Strength Cond Res 37(9): 1870-1876, 2023-Strategic limb occlusion applied after exercise may facilitate recovery, not only in directly targeted tissue but also in remote areas of the body. The purpose of this study was to determine if postexercise ischemic conditioning (PEIC) applied directly to one leg facilitated recovery in the targeted leg and the contralateral leg that did not receive direct PEIC. Twenty active men participated in a single-blind, randomized, crossover design. Subjects completed 2 paired testing sessions (PEIC and control-SHAM) that included pre-assessments and 24-hour postassessments. Each paired testing session included an eccentric drop jump task, which has been shown to increase lower-body muscle soreness and decrease strength. After each drop jump task, occlusion cuffs were immediately applied. In the PEIC session, ∼198 mm Hg was applied directly to one leg (PEIC-Direct), whereas the contralateral leg received a nonphysiological stimuli of 20 mm Hg (PEIC-Remote). In the control-SHAM session, both legs directly and remotely received the 20 mm Hg pressure. Unilateral pre-assessments and 24-hour postassessments included muscle soreness using a visual analog scale and strength via peak torque assessment across the force-velocity spectrum (flexion/extension 60/60, 120/120, 180/180, 240/240, 300/300 °·s -1 ), and a maximal eccentric extension (30/30 °·s -1 ). Muscle soreness was significantly increased ( p < 0.05) at 24 hours compared with pretreatment except for PEIC-Direct (1.19 ± 0.78 vs. 2.32 ± 1.48, p = 0.096). Across the force-velocity spectrum, there were no significant differences observed between any associated pretest and posttest ( p > 0.05). PEIC applied directly to target leg after eccentric drop jumps attenuated perceived quadriceps muscle soreness 24 hours post; however, there was no effect on muscle strength.

Integrin ß1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells.

The expression of the angiotensin-converting enzyme 2 (ACE2) is altered in multiple chronic kidney diseases like hypertension and renal fibrosis, where the signaling from the basal membrane proteins is critical for the development and progression of the various pathologies. Integrins are heterodimeric cell surface receptors that have important roles in the progression of these chronic kidney diseases by altering various cell signaling pathways in response to changes in the basement membrane proteins. It is unclear whether integrin or integrin-mediated signaling affects the ACE2 expression in the kidney. The current study tests the hypothesis that integrin ß1 regulates the expression of ACE2 in kidney epithelial cells. The role of integrin ß1 in ACE2 expression in renal epithelial cells was investigated by shRNA-mediated knockdown and pharmacological inhibition. In vivo studies were carried out using epithelial cell-specific deletion of integrin ß1 in the kidneys. Deletion of integrin ß1 from the mouse renal epithelial cells reduced the expression of ACE2 in the kidney. Furthermore, the downregulation of integrin ß1 using shRNA decreased ACE2 expression in human renal epithelial cells. ACE2 expression levels were also decreased in renal epithelial cells and cancer cells when treated with an integrin α2ß1 antagonist, BTT 3033. SARS-CoV-2 viral entry to human renal epithelial cells and cancer cells was also inhibited by BTT 3033. This study demonstrates that integrin ß1 positively regulates the expression of ACE2, which is required for the entry of SARS-CoV-2 into kidney cells.

Reversal of aging-associated increase in myelopoiesis and expression of alarmins by angiotensin-(1-7).

Aging is associated with chronic systemic inflammation largely due to increased myelopoiesis, which in turn increases risk for vascular disease. We have previously shown evidence for the therapeutic potential of Angiotensin-(1-7) (Ang-(1-7)) in reversing vasoreparative dysfunction in aging. This study tested the hypothesis that ischemic vascular repair in aging by Ang-(1-7) involves attenuation of myelopoietic potential in the bone marrow and decreased mobilization of inflammatory cells. Young or Old male mice of age 3-4 and 22-24 months, respectively, received Ang-(1-7) (1 µg/kg/min, s.c.) for four weeks. Myelopoiesis was evaluated in the bone marrow (BM) cells by carrying out the colony forming unit (CFU-GM) assay followed by flow cytometry of monocyte-macrophages. Expression of pro-myelopoietic factors and alarmins in the hematopoietic progenitor-enriched BM cells was evaluated. Hindlimb ischemia (HLI) was induced by femoral ligation, and mobilization of monocytes into the blood stream was determined. Blood flow recovery was monitored by Laser Doppler imaging and infiltration of inflammatory cells was evaluated by immunohistochemistry. BM cells from Old mice generated a higher number of monocytes (Ly6G-CD11b+Ly6Chi) and M1 macrophages (Ly6ChiF4/80+) compared to that of Young, which was reversed by Ang-(1-7). Gene expression of selected myelopoietic factors, alarmins (S100A8, S100A9, S100A14 and HMGb1) and the receptor for alarmins, RAGE, was higher in the Old hematopoietic progenitor-enriched BM cells compared to the Young. Increased expressions of these factors were decreased by Ang-(1-7). Ischemia-induced mobilization of monocytes was higher in Old mice with decreased blood flow recovery and increased infiltration of monocyte-macrophages compared to the Young, all of which were reversed by Ang-(1-7). Enhanced ischemic vascular repair by Ang-(1-7) in aging is largely by decreasing the generation and recruitment of inflammatory monocyte-macrophages to the areas of ischemic injury. This is associated with decreased alarmin signaling in the BM-hematopoietic progenitor cells.

Transforming growth factor-ß1/Thrombospondin-1/CD47 axis mediates dysfunction in CD34+ cells derived from diabetic older adults.

Aging with diabetes is associated with impaired vasoprotective functions and decreased nitric oxide (NO) generation in CD34+ cells. Transforming growth factor- ß1 (TGF-ß1) is known to regulate hematopoietic functions. This study tested the hypothesis that transforming growth factor- ß1 (TGF-ß1) is upregulated in diabetic CD34+ cells and impairs NO generation via thrombospondin-1 (TSP-1)/CD47/NO pathway. CD34+ cells from nondiabetic (ND) (n=58) or diabetic older adults (DB) (both type 1 and type 2) (n=62) were isolated from peripheral blood. TGF-ß1 was silenced by using an antisense delivered as phosphorodiamidate morpholino oligomer (PMO-TGF-ß1). Migration and proliferation in response to stromal-derived factor-1α (SDF-1α) were evaluated. NO generation and eNOS phosphorylation were determined by flow cytometry. CD34+ cells from older, but not younger, diabetics have higher expression of TGF-ß1 compared to that observed in cells derived from healthy individuals (P<0.05, n=14). TSP-1 expression was higher (n=11) in DB compared to ND cells. TGFß1-PMO decreased the secretion of TGF-ß1, which was accompanied with decreased TSP-1 expression. Impaired proliferation, migration and NO generation in response to SDF-1α in DB cells were reversed by TGF-ß1-PMO (n=6). TSP-1 inhibited migration and proliferation of nondiabetic CD34+ cells that was reversed by CD47-siRNA, which also restored these responses in diabetic CD34+ cells. TSP-1 opposed SDF-1α-induced eNOS phosphorylation at Ser1177 that was reversed by CD47-siRNA. These results infer that increased TGF-ß1 expression in CD34+ cells induces dysfunction in CD34+ cells from diabetic older adults via TSP-1/CD47-dependent inhibition of NO generation.

Early onset of aging phenotype in vascular repair by Mas receptor deficiency.

Aging is associated with impaired vascular repair following ischemic insult, largely due to reparative dysfunctions of progenitor cells. Activation of Mas receptor (MasR) was shown to reverse aging-associated vasoreparative dysfunction. This study tested the impact of MasR-deficiency on mobilization and vasoreparative functions with aging. Wild type (WT) or MasR-deficient mice (MasR-/- or MasR+/-) at 12-14 weeks (young) or middle age (11-12 months) (MA) were used in the study. Mobilization of lineage-negative, Sca-1-positive cKit-positive (LSK) cells in response to G-CSF or plerixafor was determined. Hindlimb ischemia (HLI) was induced by femoral artery ligation. Mobilization and blood flow recovery were monitored post-HLI. Radiation chimeras were made by lethal irradiation of WT or MasR-/- mice followed by administration of bone marrow cells from MasR-/- or WT mice, respectively. Nitric oxide (NO) generation by stromal-derived factor-1α (SDF) and mitochondrial reactive oxygen species (mitoROS) levels were determined by flow cytometry. Effect of A779 treatment on mobilization, blood flow recovery, and NO and ROS levels were determined in young WT and MasR+/- mice. Circulating LSK cells in basal or in response to plerixafor or G-CSF or in response to ischemic injury were lower in MasR-/- mice compared to the WT. Responses in MasR+/- mice were similar to the WT at young age but at the middle age, impairments were observed. Impaired mobilization to ischemia or G-CSF was rescued in WT → MasR-/- chimeras. NO levels were lower and mitoROS were higher in MasR-/- LSK cells compared to WT cells. A779 precipitated dysfunctions in young-MasR+/- mice similar to that observed in MA-MasR+/-, and this accompanied decreased NO generation by SDF and enhanced mitoROS levels. This study shows that mice at MA do not exhibit vasoreparative dysfunction. Either partial or total loss of MasR precipitates advanced-aging phenotype likely due to lack of NO and oxidative stress.

Integrin ß1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-ß Receptor-2.

The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin ß1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin ß1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-ß receptor 2. These results unravel a new mechanism of integrin ß1 in tumor growth by modifying the expression of kindlin-2 and TGF-ß receptor 2 signaling.

ACE2 gene transfer ameliorates vasoreparative dysfunction in CD34+ cells derived from diabetic older adults.

Diabetes increases the risk for ischemic vascular diseases, which is further elevated in older adults. Bone marrow-derived hematopoietic CD34+ stem/progenitor cells have the potential of revascularization; however, diabetes attenuates vasoreparative functions. Angiotensin-converting enzyme 2 (ACE2) is the vasoprotective enzyme of renin-angiotensin system in contrast with the canonical angiotensin-converting enzyme (ACE). The present study tested the hypothesis that diabetic dysfunction is associated with ACE2/ACE imbalance in hematopoietic stem/progenitor cells (HSPCs) and that increasing ACE2 expression would restore reparative functions. Blood samples from male and female diabetic (n=71) or nondiabetic (n=62) individuals were obtained and CD34+ cells were enumerated by flow cytometry. ACE and ACE2 enzyme activities were determined in cell lysates. Lentiviral (LV) approach was used to increase the expression of soluble ACE2 protein. Cells from diabetic older adults (DB) or nondiabetic individuals (Control) were evaluated for their ability to stimulate revascularization in a mouse model of hindlimb ischemia (HLI). DB cells attenuated the recovery of blood flow to ischemic areas in nondiabetic mice compared with that observed with Control cells. Administration of DB cells modified with LV-ACE2 resulted in complete restoration of blood flow. HLI in diabetic mice resulted in poor recovery with amputations, which was not reversed by either Control or DB cells. LV-ACE2 modification of Control or DB cells resulted in blood flow recovery in diabetic mice. In vitro treatment with Ang-(1-7) modified paracrine profile in diabetic CD34+ cells. The present study suggests that vasoreparative dysfunction in CD34+ cells from diabetic older adults is associated with ACE2/ACE imbalance and that increased ACE2 expression enhances the revascularization potential.

Targeting Angiotensin-Converting Enzyme-2/Angiotensin-(1-7)/Mas Receptor Axis in the Vascular Progenitor Cells for Cardiovascular Diseases.

Bone marrow-derived hematopoietic stem/progenitor cells are vasculogenic and play an important role in endothelial health and vascular homeostasis by participating in postnatal vasculogenesis. Progenitor cells are mobilized from bone marrow niches in response to remote ischemic injury and migrate to the areas of damage and stimulate revascularization largely by paracrine activation of angiogenic functions in the peri-ischemic vasculature. This innate vasoprotective mechanism is impaired in certain chronic clinical conditions, which leads to the development of cardiovascular complications. Members of the renin-angiotensin system-angiotensin-converting enzymes (ACEs) ACE and ACE2, angiotensin II (Ang II), Ang-(1-7), and receptors AT1 and Mas-are expressed in vasculogenic progenitor cells derived from humans and rodents. Ang-(1-7), generated by ACE2, is known to produce cardiovascular protective effects by acting on Mas receptor and is considered as a counter-regulatory mechanism to the detrimental effects of Ang II. Evidence has now been accumulating in support of the activation of the ACE2/Ang-(1-7)/Mas receptor pathway by pharmacologic or molecular maneuvers, which stimulates mobilization of progenitor cells from bone marrow, migration to areas of vascular damage, and revascularization of ischemic areas in pathologic conditions. This minireview summarizes recent studies that have enhanced our understanding of the physiology and pharmacology of vasoprotective axis in bone marrow-derived progenitor cells in health and disease. SIGNIFICANCE STATEMENT: Hematopoietic stem progenitor cells (HSPCs) stimulate revascularization of ischemic areas. However, the reparative potential is diminished in certain chronic clinical conditions, leading to the development of cardiovascular diseases. ACE2 and Mas receptor are key members of the alternative axis of the renin-angiotensin system and are expressed in HSPCs. Accumulating evidence points to activation of ACE2 or Mas receptor as a promising approach for restoring the reparative potential, thereby preventing the development of ischemic vascular diseases.

Blood flow restriction exercise stimulates mobilization of hematopoietic stem/progenitor cells and increases the circulating ACE2 levels in healthy adults.

Adult CD34+ hematopoietic stem/progenitor cells (HSPC) in the systemic circulation are bone marrow-derived and have the propensity of maintaining cardiovascular health. Activation of angiotensin-converting enzyme-2 (ACE2)-angiotensin-(1-7)-Mas receptor pathway, the vascular protective axis of the renin-angiotensin system (RAS), stimulates vasculogenic functions of HSPCs. In a previous study, exposure to hypoxia increased the expressions of ACE2 and Mas, and stimulated ACE2 shedding. The current study tested if blood flow restriction exercise (BFR)-induced regional hypoxia recapitulates the in vitro observations in healthy adults. Hypoxia was induced by 80% limb occlusion pressure (LOP) via inflation cuff. Muscle oxygen saturation was determined using near-infrared spectroscopy. Peripheral blood was collected 30 min after quiet sitting (control) or after BFR. Lin-CD45lowCD34+ HSPCs were enumerated by flow cytometry, and ACE and ACE2 activities were determined in plasma and cell lysates and supernatants. Regional hypoxia resulted in muscle oxygen saturation of 17.5% compared with 49.7% in the control condition (P < 0.0001, n = 9). Circulating HSPCs were increased following BFR (834.8 ± 62.1/mL) compared with control (365 ± 59, P < 0.001, n = 7), which was associated with increased stromal-derived factor 1α and vascular endothelial growth factor receptor levels by four- and threefold, respectively (P < 0.001). ACE2 activity was increased in the whole cell lysates of HSPCs, resulting in an ACE2-to-ACE ratio of 11.7 ± 0.5 in BFR vs 9.1 ± 0.9 in control (P < 0.05). Cell supernatants have threefold increase in the ACE2-to-ACE ratio following BFR compared with control (P < 0.001). Collectively, these findings provide strong evidence for the upregulation of ACE2 by acute regional hypoxia in vivo. Hypoxic exercise regimens appear to be promising means of enhancing vascular regenerative capacity.NEW & NOTEWORTHY Although many studies have explored the mechanisms of skeletal muscle growth and adaptation with hypoxia exercise interventions, less attention has been given to the potential for vascular adaptation and regenerative capacity. This study shows for the first time an acute upregulation of the angiotensin-converting enzyme 2 and increase in CD34+ vasculogenic cells following an acute bout of blood flow restriction with low-intensity exercise. These rapid changes collectively promote skeletal muscle angiogenesis. Therefore, this study supports the potential of hypoxic exercise interventions with low intensity for vascular and muscle health.

Diabetic pre-programming of myelopoiesis impairs tissue repair.

Bone marrow-derived monocyte-macrophages promote healing of injured tissue cooperatively with vasculogenic hematopoietic stem/progenitor cells. However, diabetes dysregulates hematopoiesis and attenuates bone marrow-derived tissue-reparative responses. In a recent issue of The Journal of Pathology, Barman et al extensively characterized myelopoietic responses in bone marrow following skin wounding in a type 2 model of diabetes. The study demonstrated that accumulation of monocyte-macrophages in the peripheral tissues is increased due to diabetic myelopoiesis that would oppose the reparative process following tissue injury. Interestingly, in this model, pathological myelopoiesis is independent of IL-1ß. The potential prophylactic and therapeutic implications of these data are discussed in terms of paracrine signaling, macrophage polarization, and hematopoietic stem cell mobilization/retention. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A PharmD program curricular approach to addressing the opioid crisis.

BACKGROUND AND PURPOSE: The opioid crisis in the United States is widespread and requires large scale efforts to reduce the problem. A recent call by the American Association of Colleges of Pharmacy requested commitments by member schools to enact curricular changes in order to prepare pharmacy graduates to be impactful in addressing the opioid crisis. EDUCATIONAL ACTIVITY: A taskforce reviewed existing pain management and opioid-related curricular content in the North Dakota State University School of Pharmacy (NDSU SOP). As a result, all pre-existing opioid-related content was maintained and six new educational activities were designed and implemented to enhance hands-on experience and application of pain management and opioid-related concepts and skills. Students were surveyed after completing the pre-APPE curriculum regarding their confidence in identifying inappropriate use of opioids and engaging in conversations with patients about safe opioid use. FINDINGS: The full longitudinal approach to addressing pain management and opioid content in the North Dakota State University School of Pharmacy program is described. Curricular gap analysis, thoughtful coordination, faculty collaboration, and curriculum mapping were required in order to bring the full pain management and opioid curricular plan to fruition. The vast majority of students were confident in their ability to counsel patients on appropriate options for safe use and disposal of opioids (96%) and their ability to determine if a patient is at risk for opioid misuse (92%). A lower percentage (71%) were confident in their ability to converse with patients regarding opioid misuse concerns. SUMMARY: This is the first report to summarize a longitudinal, curriculum-wide approach taken to address the opioid crisis in the United States and in response to AACP's call to action for pharmacy schools. This description may be valuable to other pharmacy and health professions educational programs in search of means to implement and/or enhance their current curricular offerings related to pain management and opioids.

Hypoxic regulation of angiotensin-converting enzyme 2 and Mas receptor in human CD34+ cells.

CD34+ hematopoietic stem/progenitor cells (HSPCs) are vasculogenic and hypoxia is a strong stimulus for the vasoreparative functions of these cells. Angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7)/Mas receptor (MasR) pathway stimulates vasoprotective functions of CD34+ cells. This study tested if ACE2 and MasR are involved in the hypoxic stimulation of CD34+ cells. Cells were isolated from circulating mononuclear cells derived from healthy subjects (n = 46) and were exposed to normoxia (20% O2 ) or hypoxia (1% O2 ). Luciferase reporter assays were carried out in cells transduced with lentivirus carrying ACE2- or MasR- or a scramble-3'-untranslated region gene with a firefly luciferase reporter. Expressions or activities of ACE, angiotensin receptor Type 1 (AT1R), ACE2, and MasR were determined. In vitro observations were verified in HSPCs derived from mice undergoing hindlimb ischemia (HLI). In vitro exposure to hypoxia-increased proliferation and migration of CD34+ cells in basal conditions or in response to vascular endothelial growth factor (VEGF) or stromal-derived factor 1α (SDF) compared with normoxia. Expression of ACE2 or MasR was increased relative to normoxia while ACE or AT1R expressions were unaltered. Luciferase activity was increased by hypoxia in cells transfected with the luciferase reporter plasmids coding for the ACE2- or MasR promoters relatively to the control. The effects of hypoxia were mimicked by VEGF or SDF under normoxia. Hypoxia-induced ADAM17-dependent shedding of functional ACE2 fragments. In mice undergoing HLI, increased expression/activity of ACE2 and MasR were observed in the circulating HSPCs. This study provides compelling evidence for the hypoxic upregulation of ACE2 and MasR in CD34+ cells, which likely contributes to vascular repair.

Mitochondrial depolarization stimulates vascular repair-relevant functions of CD34+ cells via reactive oxygen species-induced nitric oxide generation.

BACKGROUND AND PURPOSE: CD34+ haematopoietic stem/progenitor cells have revascularization potential and are now being tested for the treatment of ischaemic vascular diseases in clinical trials. We tested the hypothesis that mitochondrial depolarization stimulates the reparative functions of CD34+ cells. EXPERIMENTAL APPROACH: Peripheral blood was obtained from healthy individuals (n = 63), and mononuclear cells (MNCs) were separated. MNCs were enriched for lineage negative cells, followed by isolation of CD34+ cells. Vascular repair-relevant functions of CD34+ cells, proliferation and migration, were evaluated in the presence and absence of diazoxide. Mitochondrial membrane potential, ROS and NO levels were evaluated by flow cytometry by using JC-1, mitoSOX and DAF-FM respectively. KEY RESULTS: Diazoxide stimulated the proliferation and migration of CD34+ cells that were comparable to the responses induced by stromal-derived factor-1α (SDF) or VEGF. Effects of diazoxide were blocked by either 5-hydroxydecanoate (5HD), a selective mitochondrial ATP-sensitive potassium channel (mitoKATP ) inhibitor, or by L-NAME. Diazoxide induced mitochondrial depolarization, and NO and cGMP generation that were 5HD-sensitive. The generation of NO and cGMP by diazoxide was blocked by an endothelial NOS (eNOS)-selective inhibitor, NIO, but not by a neuronal (n)NOS-selective inhibitor, Nω -propyl-L-arginine (NPA). A Ca2+ chelator, BAPTA, Akt inhibitor, triciribine, or PI3K inhibitor, LY294002, inhibited the NO release induced by diazoxide. Phosphorylation of eNOS at Ser1177 and dephosphorylation at Thr495 were increased. Diazoxide-induced ROS generation and phosphorylation of eNOS at Ser1177 were reduced by NPA. CONCLUSION AND IMPLICATIONS: Diazoxide stimulates vascular repair-relevant functions of CD34+ cells via the mitoKATP -dependent release of NO and ROS. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.

Methods for Studying the Role of RAAS in the Modulation of Vascular Repair-Relevant Functions of Stem/Progenitor Cells.

In recent years, previously unknown functions have been conferred to the RAAS and have been explored in mechanistic studies and disease models. Implication of bone marrow stem/progenitor cells in the cardiovascular protective or detrimental effects of RAAS is a prominent advancement because of the translational significance. Selected members of RAAS are now known to modulate migration, proliferation, and mobilization of bone marrow cells in response to ischemic insult, which are sensitive indicators of vascular repair-relevant functions. In this Chapter, protocols for most frequently used, in vitro, ex vivo, and in vivo assays to explore the potential of RAAS members to stimulate vascular repair-relevant functions of bone marrow stem/progenitor cells of human and murine origin.

Reversal of Bone Marrow Mobilopathy and Enhanced Vascular Repair by Angiotensin-(1-7) in Diabetes.

The angiotensin (ANG)-(1-7)/Mas receptor (MasR) pathway activates vascular repair-relevant functions of bone marrow progenitor cells. We tested the effects of ANG-(1-7) on mobilization and vasoreparative functions of progenitor cells that are impaired in diabetes. The study was performed in streptozotocin-induced diabetic (db/db) mice. Diabetes resulted in a decreased number of Lineage-Sca-1+c-Kit+ (LSK) cells in the circulation, which was normalized by ANG-(1-7). Diabetes-induced depletion of LSK cells in the bone marrow was reversed by ANG-(1-7). ρ-Kinase (ROCK) activity was increased specifically in bone marrow LSK cells by ANG-(1-7) in diabetes, and the beneficial effects of ANG-(1-7) were prevented by fasudil. ANG-(1-7) increased Slit3 levels in the bone marrow supernatants, which activated ROCK in LSK cells and sensitized them for stromal-derived factor-1α (SDF)-induced migration. Diabetes prevented the mobilization of LSK cells in response to ischemia and impaired the recovery of blood flow, both of which were reversed by ANG-(1-7) in both models of diabetes. Genetic ablation of MasR prevented ischemia-induced mobilization of LSK cells and impaired blood flow recovery, which was associated with decreased proliferation and migration of LSK cells in response to SDF or vascular endothelial growth factor. These results suggest that MasR is a promising target for the treatment of diabetic bone marrow mobilopathy and vascular disease.