Persistent vascular injury and degeneration in diabetes are attributed in part to defective reparatory function of angiogenic cells. Our recent work implicates endoplasmic reticulum (ER) stress in high-glucose-induced bone marrow (BM) progenitor dysfunction. Herein, we investigated the in vivo role of ER stress in angiogenic abnormalities of streptozotocin-induced diabetic mice. Our data demonstrate that ER stress markers and inﬂammatory gene expression in BM mononuclear cells and hematopoietic progenitor cells increase dynamically with disease progression. Increased CHOP and cleaved caspase 3 levels were observed in BM-derived early outgrowth cells (EOCs) after 3 months of diabetes. Inhibition of ER stress by ex vivo or in vivo chemical chaperone treatment signiﬁcantly improved the generation and migration of diabetic EOCs while reducing apoptosis of these cells. Chemical chaperone treatment also increased the number of circulating angiogenic cells in peripheral blood, alleviated BM pathology, and enhanced retinal vascular repair following ischemia/reperfusion in diabetic mice. Mechanistically, knockdown of CHOP alleviated high-glucose-induced EOC dysfunction and mitigated apoptosis, suggesting a pivotal role of CHOP in mediating ER stress-associated angiogenic cell injury in diabetes. Together, our study suggests that targeting ER signaling may provide a promising and novel approach to enhancing angiogenic function in diabetes. Introduction circulation and subsequently migrate into injured tissues . Diabetic retinopathy (DR) is a sight-threatening com- In diabetes, this process is hampered, resulting in reduced plication of diabetes affecting around 93 million people numbers of circulating angiogenic cells (CACs) in diabetic 1 4 worldwide . Early clinical features of DR include vascular patients . Furthermore, diabetes disrupts BM homeostasis leakage and focal retinal non-perfusion due to loss of increasing the production of pro-inﬂammatory mono- capillaries . Accumulative endothelial injury and failure to cytes, which in turn exacerbates retinal inﬂammation and repair damaged blood vessels contribute to progressive vascular degeneration . vascular degeneration and ischemia leading to advanced While the mechanisms underlying the angiogenic DR. Upon tissue injury, bone marrow (BM)-derived abnormalities in diabetes are complex, our recent work angiogenic progenitors are released from the BM into suggests that disturbance of endoplasmic reticulum (ER) is potentially involved in diabetic injury of angiogenic progenitors . The ER is one of the major organelles responsible for protein biosynthesis, protein folding and Correspondence: Sarah X. Zhang (firstname.lastname@example.org) Department of Ophthalmology and Ross Eye Institute, University at Buffalo, maturation, as well as protein trafﬁcking. Dysfunction of State University of New York, Buffalo, NY, USA the ER leads to ER stress that activates the unfolded SUNY Eye Institute, State University of New York, New York, NY, USA protein response (UPR) to maintain protein homeostasis Full list of author information is available at the end of the article Edited by M. Piacentini © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Bhatta et al. Cell Death and Disease (2018) 9:467 Page 2 of 14 Fig. 1 Dynamic changes in levels of bone marrow (BM) progenitor cells and decrease of CACs in diabetic mice. Mononuclear cells from peripheral blood or the BM of diabetic (DM) mice at 1, 3, 6, and 9 months after the onset of diabetes and their respective non-diabetic (NDM) controls + + + − were separated by Ficoll gradient centrifugation. a Numbers of CACs (Flk-1 /Sca-1 /CD34 ) in the peripheral blood. b Numbers of BM-HSCs (Lin /c- + + + + + + Kit /Sca-1 /CD34 ). c Numbers of BM-CACs (Flk-1 /Sca-1 /CD34 ; n = 3–6 mice per group). All data are presented as mean ± SD. *P < 0.05, ***P < 0.001 vs. age-matched NDM control mice (two-way ANOVA with bonferroni post hoc test) in normal cells (adaptive UPR) or promote apoptosis of release is at Zeitgeber time (ZT)-3 or ZT-5 in non- 7–10 17,18 overstressed cells (terminal UPR) . In diabetes, diabetic rats or mice, respectively . Thus, for CAC 11,12 increased ER stress is observed in a variety of tissues analysis, we collected peripheral blood and BM tissues as well as in angiogenic progenitors . Inhibition of ER from all the animals at around this time point. Consistent 15,19,20 stress signiﬁcantly enhanced the survival and function of with previous reports , we observed a signiﬁcant + + + angiogenic progenitors cultured in high-glucose (HG) decrease in CACs (Flk-1 /Sca-1 /CD34 cells) in mice conditions. These ﬁndings provide preliminary evidence after 3 months and 6 months of diabetes (Fig. 1a). Inter- that ER stress plays a causal role in diabetes-related estingly, there is no signiﬁcant difference in CACs from angiogenic dysfunction. 9-month-diabetic mice and non-diabetic controls. Yet, the Herein, we characterized the temporal development of absolute percentage of CACs was drastically reduced ER stress in BM progenitors and examined the in vivo role compared to younger mice, suggesting a potential effect of of ER stress in angiogenic progenitor dysfunction in a type aging on CACs (Fig. 1a). 1 diabetes model. Our data demonstrate that there is While reduced CACs may suggest impaired mobiliza- increased ER stress and altered UPR signaling in BM tion of progenitors, it is also possible that the production progenitors during diabetes progression. Inhibiting ER of progenitor cells is curtailed in the BM. To test this stress by chemical chaperone treatment ex vivo or in vivo possibility, we quantiﬁed BM hematopoietic stem cells signiﬁcantly mitigates diabetes-induced BM pathology, (HSCs) and BM-CACs in mice with 1, 3, 6, or 9 months of enhances angiogenic progenitor function, and promotes diabetes and age- and sex-matched non-diabetic controls. vascular repair in diabetic mice. Knockdown of Chop also We found that the number of HSCs in the BM increased improves angiogenic progenitor survival and function. by 33% at 3 months after diabetes (Fig. 1b). BM-CACs + + + These ﬁndings suggest that modulating ER stress may (Flk-1 /Sca-1 /CD34 cells) demonstrate a 1.5-fold provide a novel approach to improving angiogenic func- increase after 3 months of diabetes followed by a twofold tion in diabetes. decrease after 6 months of diabetes (Fig. 1c). Furthermore, the number of BM-CACs, but not BM-HSCs, declined Results drastically with age (Fig. 1b, c). Decreased numbers of CACs in peripheral blood of diabetic mice Increased expression of ER stress markers in BMNCs and Studies have shown that CAC levels in peripheral blood c-Kit-expressing BM cells from diabetic mice are reduced in patients with type 1 and type 2 dia- Enhanced ER stress has been observed in EOCs from 4,13,14 15,16 betes as well as in diabetic mice . However, a type 2 diabetic mice and in peripheral blood mononuclear comprehensive analysis of CAC dynamics through the cells (PBMCs) from patients with diabetes and cardio- 21–23 stages of diabetes is lacking. Herein, we examined the vascular diseases . We examined ER stress and CAC levels in peripheral blood of mice with acute inﬂammation in BM mononuclear cells (BMNCs) of STZ- (1–3 months) and chronic (6–9 months) diabetes. Prior diabetic mice. We found that the expression of Atf6, a studies have demonstrated BM progenitor cell release is major UPR gene activated in response to ER stress, was regulated by circadian rhythm and the peak of CAC upregulated by over twofolds in BMNCs in 3-month- Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 3 of 14 diabetic mice; however, the expressions of Xbp1, Atf4, durations of diabetes, we performed migration assay only and Chop were not altered (Fig. 2a). After 6 months of in EOCs from 3-month-diabetic mice. As expected, dia- diabetes, both Atf6 and Xbp1 expressions were upregu- betic EOCs failed to migrate toward the chemokine vas- lated in diabetic BMNCs. A similar increase in inﬂam- cular endothelial growth factor (VEGF), and this was matory gene IL-1β was observed in BMNCs from almost completely restored by 4-PBA treatment (Fig. 4e, 3-month- and 6-month-diabetic mice (Fig. 2a). Western f). These results suggest a critical role of ER stress in diabetes-induced angiogenic progenitor dysfunction. blot analysis shows that the protein level of GRP78, an ER chaperone involved in UPR activation, was increased in BMNCs after 9 months of diabetes (Fig. 2b, c). Interest- Systemic administration of 4-PBA and TUDCA improves ingly, the protein level of ATF6 was increased after CAC release and mitigates BM pathology in diabetic mice 3 months but not 9 months of diabetes. Since BMNCs Mobilization of CACs into the circulation upon vascular is a heterogeneous population of cells consisting injury is critical for vascular repair . To examine the of not only hematopoietic cells but many other cell effect of chemical chaperones on CAC release in vivo, types , we stained BM cells with c-Kit, a marker of the 4-PBA and tauroursodeoxycholic acid (TUDCA; 40 mg/ hematopoietic progenitor cells and examined ER stress kg, twice a week) were administered via intraperitoneal protein expression in this subpopulation . We found that injection in diabetic mice at 6 weeks after diabetes onsets. expression of GRP78, ATF6, and XBP1 was signiﬁcantly The dosage was titrated not to alter the blood glucose upregulated in BM c-Kit cells (Fig. 2d, e), suggesting levels and body weights of the diabetic mice. After an increase of ER stress in diabetic hematopoietic 6 weeks of treatment, CAC levels in the peripheral blood progenitors. were enumerated by ﬂow cytometry. Compared to control mice receiving vehicle treatment, CAC levels were sig- Diabetic EOCs display increased ER stress markers niﬁcantly increased in the mice treated with 4-PBA or BM-derived EOCs are putative angiogenic progenitors, TUDCA (Fig. 5a). These results suggest that chemical which differentiate from hematopoietic progenitors chaperone treatment can improve CAC release from the in vitro. Since hematopoietic progenitors displayed an BM. To determine whether 4-PBA and TUDCA had any increase in ER stress, we speculated that EOCs from effect on HSCs in BM, we quantiﬁed the HSCs by ﬂow diabetic mice might also undergo ER stress. As shown in cytometry. As expected, HSCs were signiﬁcantly elevated Fig. 3a, there is signiﬁcant increase in expression levels of in diabetic mice as compared to non-diabetic controls, Grp78, Atf4, Chop, and Xbp1s in diabetic EOCs as which were restored to normal levels mostly with 4-PBA compared to non-diabetic EOCs. Consistent with these treatment (Fig. 5b). changes, the protein levels of CHOP and cleaved Next, we determined whether chemical chaperone treat- caspase-3 were drastically increased (Fig. 3b), suggesting ment could alleviate BM pathology. Using immunohis- that these cells are undergoing apoptosis. Moreover, the tochemistry, we examined ER stress and inﬂammation genes responsible for angiogenic function (Vegfr2, Cxcr4, markers in BM sections of diabetic and non-diabetic Mmp-9, and Adam22) and cell survival (Akt1 and Erk2) mice. We found that levels of ER chaperone GRP78 and were downregulated (Fig. 3c, d). These defects could inﬂammatory markers IL-1β and TNF-α were signiﬁcantly further escalate the damage caused by ER stress to dia- higher in diabetic BM as compared to non-diabetic control, betic EOCs. which were remarkably reduced by 4-PBA treatment (Fig. 5c, d). This suggests that chemical chaperone mitigates BM ER Inhibition of ER Stress by ex vivo chemical chaperone stress and inﬂammation, potentially improving CAC release. treatment improves the function of diabetic EOCs To determine whether ER stress plays a role in diabetes- Chemical chaperone treatment enhances angiogenic induced angiogenic dysfunction, EOCs isolated from progenitor function and survival in vivo diabetic mice or their age-matched controls were cultured To determine whether systemic chemical chaperone in the presence or absence of chemical chaperone treatment enhances angiogenic function in diabetic mice, 4-phenobutyrate (4-PBA) for 7–8 days. EOC function was EOCs from non-diabetic or diabetic mice after 6-week evaluated by counting the number of EOCs per ﬁeld and 4-PBA, TUDCA, or vehicle (PBS) treatment were counted migration assay. As shown in Fig. 4a, b, the generation of and migration assay was performed. As expected, the EOCs was unperturbed after 1 month of diabetes, sug- generation of EOCs from diabetic mice was reduced gesting that acute hyperglycemia or STZ has no effect on and these diabetic EOCs displayed reduced migration EOC function. In contrast, EOCs from 3- to 9-month toward VEGF. However, both EOC generation as well diabetic mice displayed markedly impaired EOC genera- as their migration toward VEGF was rescued by systemic tion, which was rescued by 4-PBA treatment (Fig. 4c, d). administration of 4-PBA and TUDCA in diabetic Because of cell number constraints in mice with longer mice (Fig. 6a-d). Furthermore, 4-PBA and TUDCA Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 4 of 14 Fig. 2 Increased ER stress in BMNCs and c-Kit-expressing BM cells from diabetic mice. Mononuclear cells from the BM of non-diabetic (NDM) and diabetic (DM) mice were separated by Ficoll gradient centrifugation. a mRNA levels of ER stress markers (Atf6, Xbp1s, Atf4, Chop) and Il-1β measured by qRT-PCR (n = 3 mice per group). b Protein levels of ER stress markers in BMNCs measured by western blot analysis. c Quantiﬁcation of protein levels using densitometry (n = 3 mice per group; two-way ANOVA with bonferroni post hoc test). d Flow cytometric analysis of ER stress in c-Kit-expressing BM cells (represents hematopoietic progenitor cell fraction) from 3-month-diabetic mice. e Quantiﬁcation of median ﬂuorescence intensity (MFI) as compared to NDM control (n = 3 mice per group). All data are presented as mean ± SD. *P < 0.05, **P < 0.01 vs. NDM (Student’s t-test) Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 5 of 14 Fig. 3 Expression of UPR and functional genes in EOCs. BMNCs from non-diabetic (NDM) and diabetic (DM) mice were cultured in EGM-2 medium for 7 days to generate EOCs. a Day 7 EOCs were harvested and expression of ER stress markers was determined by qRT-PCR. Data are presented as mean ± SD (n = 4 mice per group). b Protein levels of CHOP and cleaved caspase-3 (c-casp-3) in EOCs were examined by western blot analysis and quantiﬁed using densitometry (mean ± SD, n = 3 mice per group). c Expression of ER stress and functional genes in EOCs was measured by qRT-PCR (mean ± SD, n = 5–7 mice per group). d The mRNA levels of selected UPR and functional genes in EOCs assessed by PCR array (mean ± SD, n = 3 mice per group). *P < 0.05, **P < 0.01 (Student’s t-test) markedly alleviated diabetes-induced apoptosis of EOCs 4-PBA protects against retinal vascular degeneration (Fig. 6e, f). induced by ischemia reperfusion (I/R) injury in diabetic To elucidate the mechanisms underlying the protective mice effect of 4-PBA and TUDCA, we examined the expression Next, we addressed whether systemic chemical cha- of genes involved in ER stress, inﬂammation, and function perone treatment could mitigate vascular injury induced of EOCs. We found that chemical chaperones signiﬁcantly by retinal ischemia/reperfusion (I/R). This model has been downregulated ER stress marker Chop and inﬂammatory shown to develop retinal acellular capillary formation that 27,28 cytokine IL-1β while upregulating Vegfr2 (Fig. 6g). recapitulates diabetic vascular changes . Six weeks These results suggest that systemic treatment with after diabetes onsets, 4-PBA (40 mg/kg, twice a week) was chemical chaperones signiﬁcantly improves angiogenic administered intraperitoneally in diabetic mice for function, reduces inﬂammation, and enhances their 6–8 weeks. At the end of the treatment, retinal I/R was survival. induced in one eye and retinal vascular degeneration was Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 6 of 14 Fig. 4 Inhibition of ER stress by ex vivo chemical chaperone treatment enhances the functionality of diabetic EOCs. BMNCs from non- diabetic (NDM) and diabetic (DM) mice were cultured in EGM-2 medium with or without 20 μmol/l of 4-PBA for 7–8 days. a EOCs from 1-month- diabetic mice and their non-diabetic controls (white circles) were counted. b EOC quantiﬁcation after 6 days of culture (n = 3 mice per group). c EOCs were counted. d EOC quantiﬁcation after 6 days of culture (n = 3-6 mice per group). e In vitro migration assay of EOCs toward VEGF. f The migrated cells were stained with DAPI and counted. The results are expressed as fold of NDM control (four mice per group). Scale bars, 100 μm. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test (a, b) and one-way ANOVA with Tukey’s post hoc test (c–f) evaluated 14 days following I/R. Acellular capillaries, Fourteen days post I/R, diabetic mice displayed a marked + + which are devoid of endothelial cells, are identiﬁed as reduction in CACs and an increase in CXCR4 /CD11b CD31-negative, Col IV-positive vessels. We found that monocytes compared to non-diabetic controls (Fig 7c,d). there was a marked increase in acellular capillaries in 4-PBA treatment almost completely restored the numbers diabetic mice compared to non-diabetic controls (Fig. 7a, of CACs and CXCR4+/CD11b monocytes (Fig. 7c,d). b, white arrows). Treatment with 4-PBA signiﬁcantly These results suggest that chemical chaperone improves reduced acellular capillary formation following I/R injury vascular repair in diabetes possibly by increasing mobili- in diabetic animals (Fig. 7a,b). zation of CACs and reducing inﬂammation. Recent studies suggest that reduced reparative CACs, along with a corresponding increase in pro-inﬂammatory Knockdown of CHOP improves EOC function and survival 5,29 cells, contribute to insufﬁcient vascular repair . Herein, under HG conditions we examined whether the beneﬁcial effect of 4-PBA can Our data show an increase in Chop in diabetic EOCs, be attributed to improved CAC function and/or reduced which was normalized by chemical chaperone treatment. pro-inﬂammatory cells by measuring the levels of CACs To determine whether CHOP plays a role in diabetic and monocytes in peripheral blood following I/R injury. angiogenic progenitor dysfunction, EOCs from C57Bl/6J Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 7 of 14 Fig. 5 Systemic administration of chemical chaperones improves CAC release and mitigates BM pathology in diabetic mice. Two-month diabetic mice were injected with 40 mg/kg of 4-PBA, 40 mg/kg of TUDCA, or PBS as control, twice a week for 6 weeks. Mononuclear cells from the BM + + + or the peripheral blood were separated by Ficoll gradient centrifugation. a Number of peripheral blood CACs (Flk-1 /Sca-1 /CD34 ; n = 7 mice per − + + + group). b Number of BM-HSCs (Lin /c-Kit /Sca-1 /CD34 ; n = 4 mice per group). c Immunohistochemistry showing GRP78 (red), IL-β (red), and TNF- α (green) in the BM sections of non-diabetic, diabetic, and diabetic mice after PBA treatment. d Mean ﬂuorescence intensity of GRP78, IL-β, and TNF-α was quantiﬁed from four mice per group. Scale bars, 100 μm. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey’s post hoc test mice were cultured and transfected with Chop siRNA or yet the mechanisms remain elusive. In current study, we control siRNA, and then treated with 25 mmol/l HG for carried out a comprehensive analysis to assess the role of 72 h. Knockdown of Chop was evaluated by qRT-PCR ER stress in BM progenitors using a type 1 diabetes (Fig. 8a). Our results show that HG treatment signiﬁcantly model. We demonstrate a dynamic increase in ER reduced the generation of EOCs and impaired their stress accompanied by dysregulated UPR and inﬂamma- migration toward VEGF, both of which were rescued by tion in diabetic BM, BMNCs, c-Kit-expressing BM cells, CHOP knockdown (Fig. 8b-e). TUNEL assay demon- and BM-derived EOCs. Systemic treatment with chemical strated that CHOP knockdown protected EOCs from chaperones leads to a near complete reversal of angio- HG-induced apoptosis (Fig. 8f, g). These results suggest genic dysfunction, attenuation of BM pathology, and for the ﬁrst time a crucial role for CHOP in angiogenic signiﬁcant enhancement in vascular repair after retinal I/R progenitor dysfunction (Fig. 8h). in diabetic animals. Moreover, CHOP knockdown enhances EOC function and survival under HG condi- Discussion tions. These ﬁndings collectively suggest a vital role of ER It is increasingly evident that dysfunction of angiogenic stress in the pathogenesis of diabetes-induced angiogenic progenitors is a key factor in vascular complication progenitor dysfunction and defects in vascular repair 13,17,30 31 development and poor wound healing in diabetes ; (Fig. 8h). Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 8 of 14 Fig. 6 Systemic chemical chaperone treatment enhances the function and survival of angiogenic progenitor cells of diabetic mice. BMNCs from non-diabetic (NDM) and diabetic (DM) after 6-week treatment of 4-PBA, TUDCA, or vehicle were cultured in EGM-2 medium for 7–8 days. a The EOCs were counted. b EOC quantiﬁcation after 6 days of culture (n = 4–6 mice per group). c In vitro migration assay of EOCs toward VEGF. d The migrated cells were stained with DAPI and counted and the results were expressed as fold of NDM control (n = 4 mice per group). e Apoptosis determined by TUNEL assay. f Apoptosis was quantiﬁed as apoptotic cells per 1000 cells (n = 3 mice per group). g mRNA levels of Chop, Il-β, and Vegfr2 in EOCs relative to NDM (n = 4 mice per group). Scale bars, 100 μm. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA with Tukey’s post hoc test Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 9 of 14 Fig. 7 Systemic chemical chaperone treatment mitigates retinal vascular degeneration after I/R in diabetic mice. Retinal I/R was induced in diabetic (DM) mice after 6-week treatment of 4-PBA or vehicle and their non-diabetic (NDM) controls. Retinal vascular degeneration was examined and the numbers of peripheral blood CACs and inﬂammatory monocytes were determined 14 days after I/R. a Representative images of retinal ﬂat mounts stained with Col IV (green) and CD31 (Red). White arrows represent acellular capillaries. Scale bars, 50 μm. b Quantiﬁcation of acellular capillaries per ﬁeld (n = 3–5 mice per group). c The number of CACs in the peripheral blood. d The number of pro-inﬂammatory monocytes (CXCR4 + /CD11b + ) in the peripheral blood (n = 3–5 mice per group). All data are presented as mean ± SE. *P < 0.05, **P < 0.01, one-way ANOVA with Newman–Keuls multiple comparison While a plethora of studies have demonstrated that CACs and the severity of vasculopathy in type 1 diabetic 4,13,32 35 diabetic CACs are dysfunctional , a thorough char- patients , suggesting potential defects in mobilization of acterization of CACs and BM progenitors, from which CACs from the BM into circulation during diabetes. CACs differentiate, is lacking. Using the STZ model, we While ER stress was found to be increased in EOCs demonstrate a signiﬁcant increase in BM progenitors after from long-term diabetic db/db mice , it remains unknown 3 months of diabetes. This is in line with the report by when ER stress is developed in BM progenitors. We show Ferraro et al., which showed an increase in BM-HSCs in that ER stress gradually elevates in BMNCs, which is a 8-week diabetic mice . Similarly, BM-CACs increase heterogeneous population of cells comprising progenitor after 3 months of diabetes but decrease at 6 months. cell/lineage-negative fraction (2–4%) as well as differ- 36–40 Although the exact mechanism is unknown, we speculate entiated/lineage-positive cells (96–98%) . To assess that the temporary increase in BM-HSCs and CACs could ER stress only in hematopoietic progenitor cell fraction, reﬂect a compensatory response to the early vascular we stained BM cells with c-Kit and demonstrated, for the injury in diabetes, while the reduction after 6 months of ﬁrst time, increased ER stress in c-Kit-expressing BM cells diabetes may be attributed to impaired BM progenitor in early (3-month) diabetes. These results suggest that differentiation . In contrast to these changes, we hematopoietic progenitors are undergoing ER stress as observed signiﬁcant decrease in the numbers of CACs in early as 3 months with diabetes, which therefore may peripheral blood after 3 months of diabetes. These ﬁnd- impede their differentiation into CACs (Fig. 8h). Further, ings corroborate a previous report demonstrating an 3–9-month diabetic mice exhibited signiﬁcant reduction 19,20 inverse correlation between lower number of circulating in EOC generation (Fig. 4) , possibly attributed to Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 10 of 14 Fig. 8 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 11 of 14 (see ﬁgure on previous page) Fig. 8 Knockdown of CHOP enhances angiogenic progenitor cell function and survival under HG conditions. BMNCs from C57Bl/6J mice were cultured in EGM-2 medium for 7–8 days. On day 5 or 6, cells were transfected with Chop siRNA for 12 h and then treated with 25 mM HG for 3–4 days. a Efﬁciency of Chop knockdown was evaluated by qRT-PCR. b The EOCs were counted and (c) quantiﬁed after 3–4 days of HG treatment (n = 6 mice per group). Scale bars= 100 μm. d In vitro migration assay of EOCs toward VEGF. e The migrated cells were stained with DAPI and counted. The results are expressed as fold of NG control siRNA (n = 3 mice per group). Scale bars = 50 μm. f Apoptosis determined by TUNEL assay. g It was quantiﬁed as apoptotic cells per 1000 cells (n = 3 mice per group). Scale bars = 50 μm. h Schematic diagram showing activation of UPR pathways in EOCs under type 1 diabetic conditions. In type 1 diabetic conditions hematopoietic progenitor cells are undergoing ER stress, which impedes their differentiation into angiogenic progenitors/EOCs. Persistent ER stress increases ER-induced pro-apoptotic protein CHOP as well as c-caspase-3, suggesting a role for ER stress in EOC dysfunction. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey’s post hoc test 10,41,42 28 hampered differentiation . To further test this injury . Compared to non-diabetic controls, vascular hypothesis, BMNCs were treated with 4-PBA, a chemical degeneration was escalated in diabetic mice, indicated by chaperone widely used to facilitate protein folding and higher numbers of acellular capillaries, which was atte- inhibit ER stress , for the entire duration of culture. nuated by 4-PBA treatment. In addition, the number of Reduction of ER stress signiﬁcantly improved EOC gen- CACs in peripheral blood was signiﬁcantly increased eration, suggesting a causal role of ER stress in hemato- while the number of inﬂammatory monocytes was sig- poietic progenitor cell dysfunction in diabetes (Fig. 8h). In niﬁcantly reduced in 4-PBA-treated mice. These results addition, reducing ER stress also signiﬁcantly improves suggest that alleviating ER stress by chemical chaperones the migration and alleviates apoptosis of EOCs, further could promote vascular repair by enhancing CAC func- conﬁrming the role of ER stress in diabetic angiogenic tion. At this point, direct evidence is lacking to demon- progenitor dysfunction. strate that chemical chaperone treatment could increase Interestingly, similar to increased ATF4, CHOP, GRP78, CAC mobilization into injured blood vessels and/or and XBP1s activation observed in long-term type 2 dia- improve the homing capacity of CACs to the BM. This betic EOCs, we found that transcript levels of these major question needs to be addressed in future studies. UPR genes are also upregulated in type 1 diabetic EOCs. One limitation of this study lies in the use of the che- The protein levels of ER-induced pro-apoptotic protein mical chaperones 4-PBA and TUDCA, which have been CHOP as well as c-caspase-3 are also increased, sug- shown to exhibit non-ER-related effects. For example, 4- gesting that the cells are undergoing apoptosis. These PBA possesses histone deactetylase (HDAC) inhibitor results suggest that both long-term type 2 diabetic EOCs activity . However, it inhibits HDAC activity at a much and type 1 diabetic EOCs are undergoing ER stress, spe- higher concentration within the millimolar range. In one cially affecting ATF4/CHOP and IRE1/XBP1 pathways. report, 4-PBA at a concentration of 20 mmol/l inhibited These cells are more susceptible to apoptosis and display HDAC activity by ~25% . This concentration is 1000- impaired function. Inhibiting ER stress with chemical fold higher than the dose used in our study (20 µmol/l). chaperones successfully restores the function and Therefore, it is unlikely that 4-PBA protects the diabetic improves the survival of diabetic EOCs in vitro. EOCs through HDAC inhibition. Moreover, TUDCA, a Importantly, our results suggest a beneﬁcial effect of second chemical chaperone, which does not inhibit chemical chaperone treatment on improving angiogenic HDAC activity, has displayed similar effects to 4-PBA progenitor function in diabetic animals, which is due, at (Figs. 5, 6), thereby suggesting that the beneﬁcial effects least in part, to the reduction of BM pathologies. Notably, are HDAC inhibitor-independent. Future studies to elu- we designed the experiment to avoid the potential effect cidate the mechanisms of the action of these chemical of chemical chaperones on blood glucose in diabetic chaperones and to use pharmacological or genetic 8,12 mice . We show that the number of CACs in the cir- approaches for UPR manipulation to reduce ER stress in culation as well as the number of BM progenitors of angiogenic progenitor cells are warranted. diabetic mice was almost completely restored by 4-PBA Finally, our data indicate that CHOP is a potential and TUDCA treatment. Moreover, the treatment sig- molecular mediator of ER stress-associated injury of dia- niﬁcantly reduces BM pathologies, a critical factor betic angiogenic progenitors. Increased CHOP expression responsible for impaired function of angiogenic progeni- was observed in EOCs isolated from both type 1 (current 44,45 6 tors and impeded mobilization of CACs in dia- study) and type 2 diabetic mice . Knockdown of CHOP 10,46 betes . To evaluate whether reducing ER stress in signiﬁcantly improved EOC migration and prevented CACs can improve vascular repair in diabetic ischemic apoptosis induced by HG. However, it remains to be retinas, we took advantage of retinal I/R model, which tested whether CHOP inhibition could rescue CAC dys- induces vascular degeneration after an acute ischemic function in diabetic animals. The global CHOP knockout Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 12 of 14 mice are readily available and have extensively used in Early outgrowth cell culture and migration assays previous studies; however, the roles of CHOP in cell Isolation and culture of EOC were carried out as in our 49,50 6 physiology appear to be cell type- and tissue-speciﬁc . previous study . Brieﬂy, macrophage-depleted mouse Moreover, the global knockout affects multiple cell types BMNCs were plated on six-well plates coated with 5 μg/ that interact closely with each other, which would make ml human ﬁbronectin and cultured in endothelial cell the interpretation of the ﬁndings difﬁcult. Thus, the basal medium-2 supplemented with 5% FBS, VEGF-A, conditional knockout mice that lack CHOP only in ﬁbroblast growth factor, insulin-like growth factor-1, angiogenic progenitor cells would help delineate the role epidermal growth factor, ascorbic acid, and antibiotics. of CHOP in diabetes-related CAC dysfunction and vas- Non-adherent cells were removed after 4 days in culture cular degeneration in the future. In summary, our study and new medium was applied. The culture was main- provides strong evidence that enhanced ER stress and tained through days 7–9. EOCs, recognized as attaching dysregulated UPR is implicated in diabetic BM patholo- spindle-shaped cells, were used for further analyses or gies and CAC dysfunction, which in turn leads to defec- experiments. EOC counting and Migration assays were tive blood vessel repair, ultimately contributing to the performed as described previously . development of vascular damage. A better understanding of the exact roles of UPR genes in controlling angiogenic TUNEL assay progenitor function could lead to new approach to help TUNEL assay was performed using the In Situ Cell prevent and treat diabetic complications. Death Detection TMR red kit (Roche Diagnostic, India- napolis, IN, USA) . Incubation without TdT enzyme was Materials and Methods included as a negative control. Cell nuclei were stained Animal studies with DAPI and TUNEL-positive cells were counted in Male C57BL/6J mice were purchased from Jackson nine random microscopic ﬁelds and results were averaged Laboratory (Bar Harbor, ME, USA). All animal proce- from three independent experiments. dures were approved by the Institutional Animal Care and Use Committee at the University at Buffalo, State Uni- Western blotting and qRT-PCR analysis versity of New York. Diabetes was induced by ﬁve con- These assays were performed as previously described . secutive intraperitoneal injections of streptozotocin (STZ) A list of antibodies was provided in Suppl. Table 1 and at 50 mg/kg/day. Blood glucose levels were measured primer sequences for qRT-PCR can be found in Suppl. 1 week after injections and mice with blood glucose Table 2. greater than 300 mg/dl were considered diabetic. Che- mical chaperone treatment was carried out after 6 weeks Immunohistochemistry of diabetes. Brieﬂy, mice were randomly assigned to Mouse bones were ﬁxed in 4% paraformaldehyde for receive intraperitoneal injection of 4-PBA or TUDCA 3 days and then decalciﬁed in 14% EDTA for 2–3 weeks at (Calbiochem, San Diego, CA, USA) at 40 mg/kg or an 4 °C. Following sucrose gradient dehydration process, they equivalent amount of vehicle (PBS) twice a week for were embedded in optimal cutting temperature medium 6 weeks. Non-diabetic littermates received same vehicle (Sakura Finetek Inc, Torrance, CA, USA) at −80 °C. treatment. Blood glucose and body weight were measured Cryosections (20 μm) were stained with primary anti- every alternate week. bodies (Suppl. Table 1) and their corresponding Alexa Flour 488 and Alexa Flour 594-conjugated secondary Flow cytometry-assisted cell sorting analysis antibodies, and then examined under an Olympus BX53 Flow cytometry-assisted cell sorting (FAC) analyses for microscope (Olympus, Tokyo, Japan). peripheral blood mononuclear cells (PBMCs) and BM mononuclear cells (BMNCs) were performed using Retinal I/R model and quantiﬁcation of acellular retinal mouse hematopoietic and progenitor cell isolation kit (BD capillaries Biosciences, San Diego, CA, USA) as described pre- Retinal I/R was induced in anesthetized mice . Brieﬂy, viously . One million PBMCs and BMNCs were stained the anterior chamber was cannulated with a 30-gauge with antibodies to label CACs and BM progenitors at 4 °C needle connected to a reservoir of saline. Intraocular for 30 min. Flow cytometric analyses were performed with pressure (IOP) was adjusted to 80–90 mm Hg to induce LSRFortessa ﬂow cytometer (BD Biosciences) using Flow- retinal ischemia. After 60 min, the cannula was withdrawn Jo software (Tree Star Inc., Ashland, OR, USA) to to allow reperfusion. Uninjured contralateral eye served as + + + delineate CACs (Flk-1 /Sca-1 /CD34 cells) in periph- the control. Fourteen days post I/R injury the mouse eyes + + + − eral blood, BM-HSCs (Sca-1 /c-Kit /CD34 /Lin cells), were enucleated and retinas were prepared for immuno- + + + and BM-CACs (Flk-1 /Sca-1 /CD34 cells); 100,000 ﬂuorescence study of acellular capillaries using antibodies events were counted per sample. against Col IV and CD31 (Suppl. Table 1). Retinas were Ofﬁcial journal of the Cell Death Differentiation Association Bhatta et al. Cell Death and Disease (2018) 9:467 Page 13 of 14 imaged under a Zeiss LSM confocal microscope (Carl Conﬂict of interest The authors declare that they have no conﬂict of interest. Zeiss, Oberkochen, Germany). Acellular capillaries were determined as CD31-negative and Col IV-positive vessels, and the number of acellular capillaries were counted and Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in averaged in three to ﬁve visual ﬁelds per retina. published maps and institutional afﬁliations. PCR array Supplementary Information accompanies this paper at https://doi.org/ qRT-PCR array was performed as per the manu- 10.1038/s41419-018-0501-5. facturer’s instruction (SA Biosciences, QIAGEN Inc., Received: 12 December 2017 Revised: 8 February 2018 Accepted: 21 Germantown, MD). A complete list of genes was provided February 2018 in Suppl. Table 3. 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