Vascular endothelial cell (EC) dysfunction plays a key role in diabetic complications. This study discovers significant upregulation of Quaking-7 (QKI-7) in iPS cell-derived ECs when exposed to hyperglycemia, and in human iPS-ECs from diabetic patients. QKI-7 is also highly expressed in human coronary arterial ECs from diabetic donors, and on blood vessels from diabetic critical limb ischemia patients undergoing a lower-limb amputation. QKI-7 expression is tightly controlled by RNA splicing factors CUG-BP and hnRNPM through direct binding. QKI-7 upregulation is correlated with disrupted cell barrier, compromised angiogenesis and enhanced monocyte adhesion. RNA immunoprecipitation (RIP) and mRNA-decay assays reveal that QKI-7 binds and promotes mRNA degradation of downstream targets CD144, Neuroligin 1 (NLGN1), and TNF-α-stimulated gene/protein 6 (TSG-6). When hindlimb ischemia is induced in diabetic mice and QKI-7 is knocked-down in vivo in ECs, reperfusion and blood flow recovery are markedly promoted. Manipulation of QKI-7 represents a promising strategy for the treatment of diabetic vascular complications. Vascular endothelial cell (EC) dysfunction contributes to the occurrence of diabetic complications. Here the authors report that in diabetic conditions, upregulation of the RNA binding protein QKI-7 in ECs due to the imbalance of RNA splicing factors CUG-BP and hnRNPM contributes to EC dysfunction, and that in vivo QKI-7 silencing improves blood flow recovery in diabetic mice with limb ischemia. Macrovascular  and microvascular  complications are the main drivers for the morbidity and mortality related to diabetes4. Patients with diabetes are known to have a two- to fourfold increased risk of cardiovascular diseases6.To date, there are over 400 million people with diabetes worldwide and the prevalence of the disease is predicted to continue to rise8. Under diabetic conditions, vascular complications are initiated by EC dysfunction leading to abnormal angiogenesis, enhanced permeability, increased monocyte adhesion and a trend toward increased thrombogenesis and blood flow impairment5.Vascular endothelial cells (ECs) are critical for the maintenance of vessel integrity as well as regulation of angiogenesis, inflammatory infiltration, monocyte adhesion, platelet aggregation, and intercellular barrier9. These implicate the involvement of a number of signaling pathways such as PKC, PI3K/AKT, MAPK, NF-kB, and Rho/ROCK amongst others12. Hence, therapeutic strategies to restore NO levels and suppress oxidative stress hold promise to restore EC function. However, it is still very challenging to rescue diabetic EC dysfunction. A comprehensive understanding of the mechanisms underlying dysfunction of ECs in diabetes is critical to the development of more effective treatments of vascular complications.Extensive evidence from clinical studies and diabetic animal models revealed the increase of reactive oxygen species (ROS), downregulation of nitric oxide synthase (eNOS), and attenuated nitric oxide (NO) levels in vascular ECs, as important mechanisms underlying the pathogenesis of EC dysfunction14. Using well-established protocols, iPSCs can be efficiently differentiated into ECs. Diabetic patient-derived iPS-ECs thereby provide a valuable cell model for the study of diabetic EC pathophysiology and drug screening with direct clinical relevance.The advent of induced pluripotent stem cell (iPSC) reprogramming technology has made it possible to generate disease models from patient somatic cells16, with transcriptional and post-transcriptional regulation systems cooperating to finely regulate gene expression in vascular ECs. RNA-binding proteins (RBPs) play critical roles in post-transcriptional regulation through formation of ribonucleoprotein (RNP) complexes with specific RNA sequences and/or RNA structures. Recent studies have implicated RBP dysregulation in diabetic complications18. Quaking (QKI) is one of the RBPs related to diabetic cardiomyopathy and atherosclerosis. QKI belongs to the signal transduction and activation of RNA (STAR) family, featuring conserved SH2, SH3, and KH RNA-binding domains and participates in the regulation of pre-mRNA splicing, mRNA nuclear exportation, mRNA stability, protein translation, and signal transduction19.Diabetes leads to dramatic alteration of gene expression profiles in ECs, including genes related to angiogenesis, inflammation, and cell adhesionQKI transcript isoforms named QKI-5, QKI-6, and QKI-7 share identical sequences in most of their coding districts but differ at the very end of the C-terminus. All three QKI isoforms are expressed in vascular ECs, with QKI-5 being the most abundant20. QKI-5 knockout mice are embryonic lethal at E10-12.5 due to disrupted vascular development along with decreased PECAM-1 and Tie-2 expression21. Recent work in our group demonstrated that QKI-5 significantly promoted angiogenesis and enhanced blood flow recovery in experimental hindlimb ischemia. Direct binding of QKI-5 to the 3′UTR and stabilization of STAT3 mRNA was verified, which in turn stabilized VE-cadherin and activated VEGFR222. We have also recently shown that QKI-6 promotes HDAC7 splicing and benefit vascular smooth muscle cell (VSMCs) induction from iPSCs. QKI-6 overexpressing VSMCs in combination with QKI-5 overexpressing ECs significantly enhanced the formation of vascular structures with in vivo Matrigel assay, corroborating their roles to support angiogenesis23. While both QKI-5 and QKI-6 contribute positively to angiogenesis, they act in various ways in different cell types, indicating the key impacts of the unique C-terminus sequences on their functions. The third QKI isoform QKI-7 also has a specific C-terminus and its function concerning angiogenesis is still unknown. Different to the C-tail nuclear localization signal with QKI-5, QKI-7 C-terminal sequence is weakly related to SH3 domain, suggesting a different subcellular localization. In this study, QKI-7 shows predominantly cytoplasmic localization indicating that its function may not be directly involved in RNA splicing. We have discovered that QKI-7 is significantly upregulated in mouse iPSC-derived ECs (miPS-ECs) treated with high glucose and human iPS-ECs from diabetic patients. QKI-7 is also found to be highly expressed in human coronary arterial ECs isolated from diabetic donors, and on blood vessels obtained from diabetic critical limb ischemia patients undergoing a lower-limb amputation. QKI-7 binds and promotes mRNA degradation of downstream targets CD144, NLGN1, TSG6 and contributes to vascular EC dysfunction. This has set up the hypothesis that QKI-7 holds a key role in the pathogenesis of diabetic EC dysfunction. Importantly, in this study we present robust evidence that targeting QKI-7 in vivo restores EC function in diabetes based on fully-defined mechanisms, presenting a potential therapeutic route for the treatment of diabetic vascular complications.Three of the central CD144 and FLK1 increased in a time-dependent manner  supplemented with 25 ng/ml vascular endothelial growth factor (VEGF) for 2–10 days. The differentiated cells adopted a typical cobblestone-like EC morphology , there are a number of predicted binding sites for the two RNA splicing factors within the QKI-7 gene sequence within intron 6 and intron 6–exon 7 interface districts  assays. These data are shown in Supplementary Fig. QKI-7 levels diametrically to maintain homeostasis by direct binding. Under diabetic conditions, this balance appears to become disrupted due to dysregulation of CUG-BP and hnRNPM leading to QKI-7 upregulation. Interestingly, neither the QKI-7 nor the splicing factors hnRNPM and CUG-BP expression levels altered when human Fibroblasts were treated with high glucose over a period of 3 and 6 days  bipartite consensus sequence and predicted 1430 QKI RNA-binding targets25. To explore the QKI-7 RNA targets, 35 EC function-related genes from this published candidate list were screened. When QKI-7 was overexpressed in miPS-ECs, 3 genes of the 35 candidates were found to be downregulated, including CD144, TSG6, and NLGN1  with the xCELLigence system. This method measures cell adhesion using high-density gold electrode arrays in a non-invasive, label-free manner. The iPS-ECs were cultured on the E-Plate with gold electrodes on the bottom of wells which are responsible for the completion of electric circuit of the applied electric potential. The impedance reflected the cell–cell interaction of adherent cells. The RTCA software converted impedance values into the Cell Index (CI), which serves as a readout of cell adhesion. In this study, QKI-7-overexpressing iPS-ECs showed decreased CI by xCelligence assay after VEGF-induced EC barrier disruption compared with control cells (ECs overexpressing an empty vector), indicating the defective cell barrier function associated with QKI-7 high expression  were generated from blood samples of people with diabetes, age, and sex-matched non-diabetic donors, and differentiated into vascular ECs, using a fast and highly efficient approach that we have recently reported30. Those hiPS-ECs assumed a typical cobblestone monolayer morphology , led to a decrease in transcript expression of lls Fig. . These rion Fig. .NLGN1 and TSG6 are both related to vascular development, it was hypothesized that co-transfection of NLGN1 or TSG6 with QKI-7 could mitigate the anti-angiogenic impact of ectopically expressed QKI-7. QKI-7 overexpressing iPS-ECs formed fewer capillary structure with shorter master segment length and smaller meshed area in comparison to the control cells (iPS-ECs overexpressing an empty vector)  Fig. . As expeor) Fig. . QKI-7, QKI-7 expression levels significantly impeded tube formation capacity of iPS-ECs in vitro, we next investigated the impact of QKI-7 on angiogenesis in vivo. iPS-ECs overexpressing QKI-7 in parallel with control cells (iPS-ECs overexpressing an empty vector) were mixed with Matrigel and injected subcutaneously into C57BL/6 mice for 7 days. Immunohistochemistry of the harvested Matrigel plugs demonstrated that the QKI-7 overexpressing iPS-ECs formed fewer capillary structures with irregular organization compared with the control cells, corroborating the angiogenic deficiency of iPS-ECs due to QKI-7 upregulation  was performed immediately after induction of hindlimb ischemia in STZ-induced diabetic mice. PBS was also used as an additional control. After 14 days, delivery of control miPS-ECs significantly promoted the blood flow recovery of the ischemic hindlimb, as indicated by Laser Doppler imaging, however, the angiogenesis-enhancing effect of miPS-EC transplantation was substantially compromised in the QKI-7 overexpression group  shRNA Lentivirus construct containing the CD144 promoter and tagged with GFP, and PBS were used as additional controls. Compared with the NT lentivirus control, QKI-7 knockdown significantly promoted blood flow recovery of the ischemic hindlimb in diabetic mice , and TGFβ signaling37. CUG-BP is highly expressed in the pancreatic islets of diabetic mice38. In diabetic cardiomyopathy, CUG-BP overexpression was also observed and contributed to cardiac complications39. CUG-BP was found to bind to the 3′ UTRs and negatively regulate the half-lives of transcripts encoding the RBPs CELF2, Mbnl1, and Mbnl241. With this in mind, these previous reports and our current finding of QKI-7 as another CUG-BP target, it seems likely that CUG-BP acts as an important upstream factor regulating the transcription network in diabetes. Along these lines, targeting CUG-BP could therefore provide a potential strategy to treat diabetic complications.CUG-BP is a multifunctional RBP that regulates pre-mRNA alternative splicing, mRNA stability, mRNA editing, and translation25. From this published database, we selected 35 putative QKI target genes for exploration of QKI-7’s downstream effectors based on their relevance to vascular EC functions.Using the defined QRE bipartite consensus sequence NACUAAY-N1–20-UAAY 1430 mRNA target candidates of QKI proteins were identifiedQKI-7 overexpression, the expression of CD144, NLGN1, and TSG6 was downregulated, while their expression was increased with QKI-7 knockdown. Furthermore, RIP assays and mRNA stability assays verified that QKI-7 destabilized their mRNA through direct target binding. In our previous work, QKI-5 was reported to induce increased CD144 levels in iPS-ECs by stabilizing CD144 mRNA22, which is contrary to the current finding with QKI-7. All QKI family isoforms share the same KH RNA-binding domain and only differ at the C-terminus. Thus, QKI-5 and QKI-7 might compete for binding to CD144 mRNA and recruit different post-transcriptional machinery to impact mRNA turnover in an antagonistic manner. CD144 is a critical component of EC adherens junctions that regulates barrier integrity and angiogenesis42. Due to enhanced QKI-7 binding and mRNA degradation, CD144 reduction under hyperglycemic conditions may therefore contribute to defective EC barrier function as well as compromised angiogenesis.With 27. Disruption of the functional interaction between TSG6 and hyaluronan with a specific antibody severely altered ECM formation43. TSG6 has also been shown to be a potent anti-inflammatory factor. On the other hand, when TSG6 was knocked down, the anti-inflammatory effects of human mesenchymal stromal cells were abolished44. One of the underlying mechanisms is interaction of TSG6 with CD44 to suppress NF-kB signaling47. In diabetic mice, subconjunctival injections of recombinant TSG6 promoted corneal epithelial wound healing through activation of resident progenitor cells and acceleration of M2 macrophage polarization48.TSG6 is a TNFα/IL-1-inducible secretory protein and contains a hyaluronan-binding domain homologous to the N122-terminal half of CD44 and participates in the regulation of cell–cell interactions50. The current study revealed that QKI-7 controlled downregulation of TSG6 in diabetic ECs, and that THP-1 monocyte adhesion was decreased after QKI-7 knockdown and TSG6 expression level restoration, suggesting that as a potent anti-inflammatory factor, TSG6 maintains vascular EC function and inhibits atherogenesis.Atherosclerosis is one of the major diabetic complications and inflammatory events in the vascular endothelium. When harmed by oxidative stress and other factors associated with diabetes, activated ECs release cytokines and chemokines to attract monocytes that adhere and develop into macrophages and foam cells, ultimately driving atherogenesis53. In vitro, NLGN1 demonstrated abundant expression in vascular ECs and NLGN1 expression dynamics significantly affected the tube formation capacity of ECs. In vivo, NLGN1 knockout caused structural defects in the vascular tree of the developing mouse retina with disrupted cell–cell junctions and aberrant distribution of laminin, VE-cadherin and α6 integrin. Our present study is in agreement with the above reports that NLGN1 is correlated with the tube formation capacity of ECs. Further to the observed negative regulation of QKI-7, NLGN1 downregulation in ECs may therefore play an important role in the ontology of diabetic EC dysfunction.NLGN1 is a cell surface protein that was originally found to be involved in synaptic transmission through cell–cell interaction and collaboration with neurexin family members. Recently, NLGN1 was reported to play an important role in vascular development55. Our current study presents a similar result further to evaluation of hindlimb hemodynamics following induction of ischemia in diabetic mice. Interestingly, when QKI-7 was knocked down by shRNA lentivirus infection targeting endogenous ECs in diabetic mice in vivo, blood flow recovery in ischemic hindlimbs was dramatically improved. On the other hand, QKI-7 overexpression in iPS-ECs substantially inhibited their pro-angiogenic effect when transplanted in diabetic mice, indicating that QKI-7 serves an effective anti-angiogenic function. Of the three QKI-7 targets discovered in the current study, CD144 is a well-known endothelial adhesion molecule that is critical for intercellular junction and vascular development. Likewise, both TSG6 and NLGN1 participate in regulation of EC-cell–matrix interaction which is critical for vasculogenesis. Downregulation of these important angiogenesis-related factors by QKI-7 therefore partly explains its negative impact on angiogenesis. While diabetic endothelial dysfunction-related angiogenesis deficiency is multifactorial, and a number of therapeutic regimens have been investigated for the treatment of chronic ischemic disorders and diabetic wound healing, current clinical strategies are incompletely effective due to the limited specific therapeutic targets of these methodologies56. In our study, in vitro rescue assay verified that reduced QKI-7 expression ameliorated diabetic EC dysfunction, namely defective barrier formation, impaired angiogenesis, and increased adhesion of monocytes. In addition, in vivo downregulation of QKI-7 in diabetic mice exerted positive effects on angiogenesis, promoting the blood flow recovery of ischemic hindlimbs. Taken together, these findings suggest that QKI-7 may represent a potential target for the treatment of diabetic complications. Importantly, as an upstream RBP, QKI-7 has potential to work as a key negative regulator to modulate multiple effectors and to impact various aspects of EC function, including barrier integrity, angiogenesis and inflammation. Therefore, specific modulation of QKI-7 levels is likely to be superior to therapeutic strategies directed toward individual targets to reduce diabetic complications.Defective angiogenesis associated with vascular endothelial dysfunction is a shared abnormality in many diabetic complications, including ischemic cardiovascular disease and wound healing deficiencies. Failure to respond to ischemia by promoting angiogenesis is intimately correlated with poor clinical outcomes in diabetes. In STZ-induced diabetic mice, the recovery of blood flow was significantly compromised after ligation or excision of the common femoral artery with the ischemic hindlimb showing fewer collateral arteries visible on angiography25, so it is conceivable that there are more RNA-binding targets for QKI-7 under diabetic conditions. Our ongoing RIP-seq work is aimed at exploring more QKI-7 binding targets so that a more comprehensive, QKI-7-centered regulation network may be built to better understand the underlying mechanisms and identify powerful targets for diabetes treatment. Second, specific signaling pathways involved in the dysregulation of CUG-BP/hnRNPM/QKI-7 in diabetes need to be elucidated. For example, aberrant PKC signaling activation has been investigated in diabetic cardiomyopathy57 and activated PKC isozymes α/β were revealed to contribute to abnormal alternative splicing through phosphorylation and upregulation of CUG-BP/Rbfox2 proteins. Third, it is important to define how QKI isoforms are able to work distinctly via specific C-termini. The C-terminal region of QKI-7 (-EWIEMPVMPDISAH-) contains three negatively charged residues and theoretical pI of 4.13 (https://web.expasy.org/protparam/), which is quite different from that of QKI-5 (-GAVATKVRRH DMRVHPYQRI VTADRAATGN-), which contains 6 positively charged residues with theoretical pI of 11.44, and that of QKI-6 (-GMAFPTKG-), with theoretical pI of 8.75. The distinctive C-terminal composition of QKI isoforms thereby determines their difference in partner recruitment, complex formation, and influences on stability of RNA targets and functions. A possible strategy would be to design small molecules against the QKI-7 C-terminus, based on crystallographic structure elucidation, to promote development of specific and effective clinical applications.There are still some questions that need to be answered in relation to the functional features and underlying mechanisms of QKI-7 signaling. First, there are multiple mRNA transcripts containing the defined QRE bipartite consensus sequenceIn summary, the present study provides key insight into the important role of QKI-7 in the pathogenesis of EC dysfunction associated with diabetes. Imbalance of CUG-BP/hnRNPM regulation in diabetic ECs leads to upregulation of QKI-7, which in turn enhances mRNA degradation of RNA-binding targets, CD144, TSG6, and NLGN1. This subsequently contributes to diabetic endothelial dysfunction, including increased permeability, impaired tube formation and increased monocyte adhesion  in DMEM (ATCC 30-2002) supplemented with 10% fetal bovine serum (FBS) , 100 IU/ml penicillin, and 100 μg/ml streptomycin (PenStrep) (Thermo Fisher Scientific 10378-016), 10 ng/ml recombinant human leukemia inhibitory factor (Millipore LIF1010) and 0.1 mM 2-mercaptoethanol (Life Technologies 31350-010) in a humidified incubator supplemented with 5% CO2. Cells were passaged every 2 days at a ratio of 1:6. EC differentiation of iPSCs was induced by seeding the cells on type IV mouse collagen -coated dishes in differentiation medium (DM) containing α-MEM (Life Technologies 32571036) supplemented with 10% FBS (Invitrogen 10270106), 0.05 mM 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin in the presence of 25 ng/ml VEGF (Thermo Fisher Scientific PMG0111) for the time points indicated. For high-glucose treatment, culture medium was supplemented with D-glucose to reach concentrations indicated in the Figures. L-glucose was used as controls in these experiments.Mouse iPSCs were generated in our laboratory by transfection of pCAG2LMKOSimO vector expressing c-Myc, Klf4, Oct4, and Sox2 into MEFs30. Mononuclear cells from donor peripheral blood were expanded and reprogrammed into iPS cells by transfection of nonintegrating episomal plasmid vectors pEB-C5 , and pEB-Tg vector (overexpressing SV40 large T antigen). The pluripotency and germ layer differentiation capacity of the generated iPS lines were verified. Ethical approval was obtained from the Office for Research Ethics Committees of Northern Ireland (ORECNI) (REC 14/NI/1109).Diabetic patient-specific iPSCs were generated based on a protocol which we have recently reportedn = 3. The QKI-7 expression was also verified in four additional iPS-ECs lines generated from four independent (sex and age-matched) diabetic donors, based on three biological replicates with n = 3. On the day of blood sample collection, a detailed medical history was obtained and blood pressure was measured. A 20 ml sample of peripheral blood was obtained by venepuncture into VACUETTE® K3 EDTA-coated 4-ml tubes .Verbal and written information about the study was provided to all participants and written informed consent was obtained prior to study procedures from those willing and consenting to take part. Patients with type 1 and type 2 diabetes of more than 15 year standing and age- and sex-matched non-diabetic volunteers, who acted as controls, were recruited to this study. Patients unable to provide informed consent for the study were excluded. All the iPS cells-based in vitro and in vivo studies were confirmed in two independent non-diabetic and two diabetic donors sets based on three biological replicates with Human iPSCs were cultured using mTeSR-1 medium (Stemcell 85850) following the manufacturer’s instructions. For EC differentiation, iPSCs were first cultured for 3 days in a 1:1 mixture of DMEM/F-12 (Thermo Fisher Scientific 31330038) with Neurobasal medium (Thermo Fisher Scientific 21103049) supplemented with N2 (Thermo Fisher Scientific 17502048), B27 (Thermo Fisher Scientific 17504044), 8 µM CHIR99021 (Sigma-Aldrich SML1046), and 25 ng/ml BMP4 (Life Technologies PHC9534). Thereafter, the cells were cultured for 2 days in StemPro 34 medium (Thermo Fisher Scientific 10639011) supplemented with 200 ng/ml hVEGF (Life Technologies PHC9391) and 2 µM Forskolin (Sigma-Aldrich F6886). On day 6, CD144 positive cells were magnetically sorted using MicroBeads Kit (Miltenyi BIotec 130-097-857) and cultured in EGM-2 media (LONZA 00190860). Human coronary artery endothelial cells were purchased from Lonza: HCAEC Coronary Art Endo Cells (CC-2585), DHCAEC Human Coronary Art Endo Cells Diabetic (CC-2921). The cells were cultured in EGM-2 media (LONZA 00190860). All methods regarding the mouse and human iPSC culture and differentiation were performed in accordance with the relevant institutional guidelines and regulations including Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects.L-Glucose (G5500) or D-glucose (G7021), both from Sigma, were reconstituted in fibroblast media (10% FBS F-12K medium (Kaighn’s modification of Ham’s F-12 medium) by ATCC (30-200). Fibroblasts (HFL1 (ATCC® CCL-153™)) were seeded in 6-well plates in 2 ml media per well at a seeding density of 100,000 or 200,000 cells for a total of 6 and 3 days of glucose treatment, respectively. Cell culture media were substituted on day 2 and on day 4. After the initial glucose treatment on day 0, the treatments were repeated with L- or D-glucose at a final concentration of 30 mM on day 2 and day 4 until harvested for subsequent real-time PCR analysis.6 iPS-ECs were resuspended in Nucleofector® solution, mixed with 5-µM SiRNA and transferred into an amaxa certified cuvette. A scrambled SiRNA (Thermo Fisher AM4611) was used as control. After 48 h, the cells were harvested for quantification of gene and protein levels of QKI-7.The SiRNA duplex for specific silencing of QKI-7 was synthesized (Sigma-Aldrich). The SiRNA sequence was -GUGAGGAGAUUGGUAUUAGUU-. A total of 1 × 10QKI-7 ORF was cloned into pReceiver-Lv225 vector to generate QKI-7 overexpression construct (GeneCopoeia). For lentivirus packaging, 1 × 107 293-T cells were transfected with QKI-7 overexpression plasmid, pCMV-dR8.2 (Addgene 8455), pCMV-VSV-G (Addgene 8454), and Endofectin (GeneCopoeia EFM1004). The supernatant containing the lentivirus was harvested twice at 48 and 96 h later, filtered (0.45 µm) and stored at −80 °C. Overexpression constructs for hnRNPM (plasmid #64924), CUG-BP (plasmid #61276) and NLGN1 (plasmid #15260) were obtained from Addgene. TSG6 (NM_007115.3) ORF was cloned into the 3rd generation of lentivirus vector to generate TSG6 overexpression construct (VectorBuilder). 3′ UTR sequence of NLGN1 was obtained by RT-PCR and cloned downstream of luciferase (hLuc) reporter gene in a Firefly/Renilla Duo-Luciferase reporter vector pEZX-MT06 (GeneCopoeia). Plasmid transfections were performed using Endofectin following the manufacturer’s instructions.GAPDH served as the endogenous control to normalize the amounts of RNA in each sample. The primers are shown in Supplementary Table Total RNA was extracted using the RNeasy Mini Kit Qiagen 74104) according to the manufacturer’s protocol. Two micrograms of RNA were reverse transcribed into cDNA with random primer by reverse transcriptase (RT) (Thermo Fisher Scientific 4374966). Relative gene expression was determined by quantitative real-time RT-PCR, using 20 ng cDNA (relative to RNA amount) for each sample with the SYBR Green Master Mix (Thermo Fisher Scientific A25742) in a 10 μl reaction. Ct values were measured using a LightCycler 480 sequence detector (Roche). 04 accordThe concentration of TSG6 released into the supernatant of 6-day differentiated ECs was detected by TSG6 ELISA (Sigma-Aldrich RAB1092) according to the manufacturer’s procedure.Cells were harvested and washed with cold PBS, resuspended in lysis buffer  supplemented with protease inhibitors (Roche 11697498001) and lysed by ultra-sonication  (Bradson Sonifier150) to obtain whole-cell lysate. The protein concentration was determined using the Bradford Dye Reagent (Bio-Rad 500-0205). Fifty micrograms of whole lysate was applied to SDS-PAGE and transferred to Hybond PVDF membrane , followed by standard western blot procedure.6 single cells were resuspended in 100 μl FACS buffer (PBS supplemented with 10% FBS) and stained by incubation with 5 μl CD144-APC antibody for 30 min at 4 °C in the dark. The cells were washed twice with PBS before resuspension in 1 ml PBS for FACS experiment on an Attune NxT Flow Cytometer and data were analyzed with Attune software (Thermo Fisher Scientific).iPSCs derived ECs were dissociated by incubation with 0.05% Trypsin-EDTA (Thermo Fisher Scientific 25300054) for 5 min at 37 °C. A total of 1 × 10The bound primary antibodies were detected by the use of horseradish peroxidase (HRP)-conjugated secondary antibodies by Bio-Rad  and the ECL detection system . Primary antibodies include QKI-7 , CD144 , CD31 , KDR , FLK1 , eNOS , NLGN1 , β-actin , and ZO-1 . For immunofluorescence staining, Alexa Fluor secondary antibodies were used . For flow cytometry, CD144-APC  and Mouse IgG1 kappa Isotype Control-APC  were used.4/well of iPS-ECs were seeded on collagen-coated wells of a 12-well plate in EGM-2 medium. 0.33 μg/well of reporter plasmids were co-transfected with QKI-7 or control (0.17 μg/well) plasmids using Endofectin Transfection Reagent (GeneCopoeia EFM1004) according to the protocol provided. pGL3-Luc Renilla (0.1 μg/well) was included in all transfection assays as an internal control. Luciferase and Renilla activity assays were detected 48 h after transfection using Dual-Glo Luciferase Assay System (Promega E2920). Relative luciferase units (RLU) was defined as the ratio of luciferase activity to Renilla activity with that of control set as 1.For luciferase reporter assays, 4 × 10QKI, CUG-BP, and hnRNPM motifs.RBP immunoprecipitation assays were performed on iPS-ECs using the Magna RIP kit from Millipore (17-700) according to the protocol provided. A QKI-7 specific antibody from Millipore, CUG-BP (PA5-85997), hnRNPM (MA1-91607) and mouse or rabbit IgG-purified antibodies were used. The precipitated RNA was subjected to RT-PCR using specific primers for the binding sites of Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked in 5% swine or donkey serum in PBS for 30 min before incubation with primary antibodies, previously mentioned, for 1 h at 37 °C. Alexa Fluor secondary antibodies by Thermo Fisher Scientific were incubated for 45 min at 37 °C. Cells were counterstained with 4′,6-diamidino-2-phenylindole , mounted in Fluoromount-G , and imaged using a fluorescent  or confocal microscope .Tissues were fixed with cold acetone (4 °C), washed with TBS and blocked in 5% donkey serum in TBS for 2 h at room temperature in a humidified atmosphere before incubating with primary antibodies overnight at 4 °C. The following day, Alexa Fluor secondary antibodies were incubated for 45 min at 37 °C and DAPI solution  for 5 min at room temperature. Tissues were mounted on VectaShield Antifade Mounting Medium (H-1000) and imaged using a fluorescent  or confocal microscope .2O for 30 s. Tissues were then placed into filtered eosin solution (Leica 3801590E) for 3 min. Tissues were next dehydrated with 75, 95, and 100% absolute ethanol. Finally, for the clearing step, tissues were rinsed with clearing agent (Leica 3803600E) for 9 min and slides were mount with DPX mountant (Sigma 06522).Tissue sections were rinsed in tap water for 5 min to hydrate and then stained with Harris’ hematoxylin solution (Leica 381560E) for 5 min at room temperature. Tissues were then rinsed in tap water for 3 min. Next, tissues were dipped in 1% acid ethanol  for 10 s and then soaked into 1% ammonia in H2O2 in TBS for 15 min at RT. Tissues were then incubated with secondary antibody  in 5% donkey serum, 1% BSA, and in 0.025% TBS-T for 45 min at 37 °C in humidity DAB solution  and Dab Substrate buffer (K4006 Lot 104224) was then applied to tissue sections for 10 min in the dark, and washed for 15 min with 0.025% TBS-T and for another 5 min rinsed with tap water. Finally, tissues were stained with haematoxylin blue as described in our H&E staining omitting the eosin incubation step.Sections were air-dried at RT for 15–20 min. Sections were next rinsed shortly with tap water and incubated for 10 min in cold 1:1 acetone:methanol solution and let them dry for 40 min at RT. Sections were then permeabilized with 0.2% Triton X-100 in TBS for 10 min at RT. And washed with 0.025% TBS-T for another 15 min. Next sections were blocked with BLOXALL® Blocking Solution (30150 ZF0726) for 10 min at RT (Peroxidase blocking) and washed with 0.025% TBS-T for another 15 min at RT. Sections were then blocked for 2 h at room temperature with 5% Donkey serum in 1% BSA in 0.025% TBS-T, and incubated overnight at 4 °C with primary antibody (aSMA Ab5694 and CD31 Ab28364). Next day, sections were washed with 2 × 5 min with TBS and blocked in 0.3% H4 iPS-ECs were seeded onto the Matrigel bed of each well and incubated at 37 °C. The formation of tube structures was observed at 6 and 8 h, which was quantified as tube length and meshed area using Image J.Hundred microliters of Matrigel (Corning 356231) was distributed into wells of a 96-well plate using pre-cooled tips and incubated at 37 °C for 1 h. A total of 1 × 102 cell culture incubator until the cell index (CI) reached plateau, which represented the impedance of electron flow caused by adherent ECs. At this stage, the culture medium was replaced by OptiMEM to starve the cells for 2 h. Culture medium was subsequently changed to normal EGM-2 supplemented with 50 ng/ml VEGF and real-time CI monitoring continued for 24 h.iPS-ECs were seeded on type IV mouse collagen -coated E-Plate VIEW 16  in EGM-2 medium containing 25 ng/ml VEGF at 40,000 cells/well. Continuous monitor was performed overnight using xCELLigence RTCA DP  in a standard COAnimals used in these studies were housed with free access to standard food and water at a room temperature of 21 ± 2 °C relative humidity of 45 ± 15% and a 12-h-light/dark cycle. All experiments were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 and approved by the Queen’s University Belfast Animal Welfare and Ethical Review Body. Work was performed under the project license number PPL2821.5 QKI-7-overexpressing iPS-ECs or control cells (iPS-ECs overexpressing an empty vector) were mixed with 150 µl Matrigel and injected subcutaneously into the back or flank of 10-week-old male C57BL/6 mice. Six injections were conducted for each group based on n = 3 biological replicates. Seven days later, the mice were sacrificed and the plugs were harvested and frozen in liquid nitrogen for cryosectioning and immunostaining. Nine plugs for each group were used for quantification.In total, 5 × 104 iPS-ECs were seeded into wells of a 96-well plate and cultured at 37 °C in 5% CO2 and 95% air humidified atmosphere. After 24 h, 1 × 104 Vybrant-labeled THP-1 cells were added into each well and co-cultured with iPS-ECs for 1 h. Thereafter, the cells were washed three times with PBS to remove the THP-1 cells in suspension. THP-1 adhesion was measured at six different areas under the microscope and the results were expressed as the number of THP-1 cells per mm2.In total, 2 × 10About 10 mg/ml STZ solution was prepared with citrate buffer at pH 4.5. Ten-week-old male C57BL/6 mice weighing more than 20 g were fasted for 4 h before intraperitoneal injection of STZ solution at 50 mg/kg (5 µl/g). Injections were performed for 5 consecutive days. One week later the blood glucose level was determined. Blood readings above 15 mmol/l were considered diabetic. All experiments were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 and approved by the Queen’s University Belfast Animal Welfare and Ethical Review Body. Work was performed under the project license number PPL2821.CD144 promoter were designed for use in the in vivo experiments to target QKI-7 in ECs. shRNA sequence can only be driven by polymerase III promoters such as the U6 or H1 promoters. Since the tissue-specific promoter is a Pol II promoter, shRNA transcription was achieved via the miR30-based method, in which shRNA was embedded in a miR30 scaffold to be transcribed as an artificial miRNA expressing cassette. Vector ID VB180427-1084hrb.shQKI-7 and shNT vectors under the 60. For EC transplantation, QKI-7 overexpressing iPS-ECs or control cells were trypsinized and 1 × 106 cells in 100 µl PBS were injected intramuscularly into the adductors of ischemic animals. For in vivo lentivirus infection, 108 transducing units (TU shRNA lentivirus) (non-targeting (NT) or QKI-7 (pLV[miR30-shRNA]-Cd144>EGFP) in a construct containing CD144 promoter, to target ECs) in 100 µl PBS was injected intramuscularly into the adductors. Tissue blood flow of both legs was sequentially assessed by Laser Doppler imaging (moorLDL2-IR) at 7 and 14 days. All experiments were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 and approved by the Queen’s University Belfast Animal Welfare and Ethical Review Body. Work was performed under the project license number PPL2821.The mouse hindlimb ischemia model was performed by ligation of femoral artery62. Sections were imaged using the Panoramic SCAN with Zeiss Plan-apochromat 20×/0.8 objective .Arterial tissue was isolated from patients with critical limb ischemia undergoing a lower-limb amputation. Patients age: 72.7+/−11.3, sex: males 87.4%, degree of atherosclerosis: ***. Arterial plaques were graded according to the Oxford grading system. All human arterial samples were obtained with informed consent and procedures were performed in accordance with institutional guidelines and the Declaration of Helsinki . The ethics application was approved by the NRES Committee North West - Lancaster and Manchester Metropolitan University Internal Ethics Approval Committee. Tissue was fixed in 4% paraformaldehyde/PBS, processed for wax embedding and cut into 7-µm sections. Immunohistochemical analysis was carried out using the QKI-7 antibody (as above), alongside a mouse non-immune IgG control, and developed using the DAB-peroxidase system (Vector Laboratories) followed by counterstaining with Mayer’s haematoxylinn = 3. Data are expressed as the mean ± SEM and were analyzed using GraphPad Prism 5 software with a two-tailed Student’s t test for two groups or ANOVA for more than two groups. A value of *p < 0.05, **p < 0.01, ***p < 0.001 was considered significant.Biological replicates were performed in all the experiments with Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The growing behavioral health needs of college students have resulted in counseling centers reporting difficulties in meeting student demand.This study aims to test the real-world voluntary use by college students of 4 digital, self-directed mental health modules based on a cognitive behavioral therapy clinical model. The findings were also compared with those of employee users.t tests (two-tailed) compared the average level of change over time on a standardized measure of clinical symptoms appropriate to each program. Cohen d statistical effect sizes were calculated for each program. Potential moderator factors  were tested together in repeated measures analysis of variance models with covariates in the full sample. Follow-up survey data (n=136) were also collected to explore user satisfaction and outcomes. Select data from another study of the same 4 programs by employee users meeting the same criteria (N=707) were examined for comparison.Archival operational data from Learn to Live were extracted for student users at 4 colleges and universities in the Midwest region of the United States (N=951). The inclusion criteria were having clinical symptoms at established levels of moderate or higher severity and the use of 2 or more of the 8 lessons of a program within a 6-month period. Unique users in each program included 347 for depression; 325 for stress, anxiety, and worry; 203 for social anxiety; and 76 for insomnia. Paired P<.001) over time were found in the mean scores for the clinical measures for each program: stress, anxiety, and worry ; insomnia ; depression ; and social anxiety . Tests of the moderating factors across programs indicated that greater improvement was strongly associated with the use of more lessons and it also differed by program, by gender , and by the use of live support (particularly coaching). Analyses of survey data found high satisfaction, improved academic outcomes, and successful integration into the university counseling ecosystem. The operational profile and outcomes of the college students were also similar to those of employee users of the same programs from our other study of employee users. Thus, this study provides a replication.The percentage of users who improved to a clinical status of no longer being at risk after program use was as follows: stress, anxiety, and worry program ; insomnia program , depression program ; and social anxiety program . Significant improvements (all Self-directed internet-based cognitive behavioral therapy mental health modules are promising as a supplement to traditional in-person counseling services provided by college counseling centers. Adjusting to college life is a significant challenge for many students , as theyA 2019 national survey of 43,140 college undergraduate and graduate students in the United States found that 42% of females and 34% of males met the criteria for moderate or severe psychological distress . For exaAlthough not a mental health disorder, sleep issues also pose risks for young adults attending colleges and universities . For exaExperiencing mental health problems can also adversely impact success at school. A recent national survey  found thThe growing behavioral health needs of college and university students have resulted in college counseling centers reporting difficulties in meeting the increased student demand ,15. ThisNew types of technology-based resources feature self-directed digital tools that are accessible from a website or mobile device. Most of these tools are asynchronous and do not involve live interaction with a mental health professional, although some are supplemented with live coaching. Many of these computerized tools are based on principles and clinical strategies derived from cognitive behavioral therapy (CBT) . The genThese reviews offer support for iCBT tools in general for being able to improve clinical outcomes, but they also call for further research to be done in real-world settings that go beyond the use of college students as convenience samples in academic research pilot studies for testing the general efficacy of tools. Few studies have used an applied study design with archival data to examine the naturally occurring experiences of voluntary users of commercially available iCBT tools for mental health issues. This emphasis on experimental over applied real-life contexts for research in this area is reflected in the literature in general and especially among investigations using college students as participants.This applied archival study was conducted with operational data from voluntary, registered users of Learn to Live, a suite of digital CBT-based self-directed programs for behavioral health issues, including anxiety, depression, social anxiety, and insomnia. The programs were hosted on a single dedicated website  and wereEach program consisted of 8 lessons that contained a brief assessment of clinical severity , videos, animations, and web-based application of CBT tools. Each set of lessons was designed to be completed in order from 1 to 8, and a prior lesson had to be completed before the user could progress to the next lesson in the program. Homework and practice with the tools was optional but recommended between lessons. Key elements of each lesson for each of the 4 programs are listed in In addition, users could opt to receive individualized coaching, and if selected, could choose the preferred channel of communication, either email, text, or telephone, with the coach. The coaches were employed by the service provider, and every coach had at least a master’s level education in psychology or counseling.teammate. These live supports are offered with the goal of adding a relational component beyond self-help as a form of activating natural social support [Users could also select a person from their personal life to communicate with during the program use, serving in a supportive role called a  support . Coachin support ,24,25.Support for these 4 programs from Learn to Live was obtained in an earlier study  that invThis study featured a longitudinal, repeated measures research design with archival data for college student users of iCBT programs offered by the same commercial provider that supported 4 different clinical issues. The goals of this study were to obtain empirical answers to the following research questions (RQs):RQ1: What is the profile among college students based on demographic factors and utilization factors for the 4 Learn to Live web-based iCBT programs?RQ2: Are each of the 4 programs effective in reducing the level of clinical symptoms after program use?RQ3: Is the extent of improvement in clinical symptoms after program use moderated by demographic factors of the user (age or gender) or by operational factors ?RQ4: Are these moderator effects (if any) similar, when tested within each of the 4 programs?RQ5: From the survey data collected following the intervention, what can be learned about the different sources of promotion of the services, the impact of use on college-related outcomes and attitudes, different aspects of the user experience with counseling, and the level of overall user satisfaction with the service?RQ6: How does the user profile and clinical outcomes of the 4 programs for college student users in this study compare with that of employee users from a different study?Learn to Live service. Users were made aware of the service as a benefit open to all students through a variety of on-campus digital and interpersonal promotional practices. There was no direct cost to the participants in this study, as access to the website with the programs was sponsored by each of the schools. Students participated voluntarily and were not paid for using the web-based tools. The study period spanned 3 years (from October 2016 through October 2019). The insomnia program was added to the service suite in November 2017 and therefore was available only for the most recent 2 years of the full study period.The data for the study were from postsecondary students using the The privacy of users was protected by having all program and survey data deidentified before being shared with the independent consultant who conducted all of the analyses. As this was an applied study of archival anonymized data collected from routine use of the service, additional informed consent from individual participants beyond their initial consent agreement in terms of use was not required. Project approval from a college or a university internal review board was also not required. This context is similar to other applied studies of commercial web-based programs ,27. The Learn To Live in the higher education market segment , (2) users were required to be at a sufficient level of clinical severity  at the start of program use, (3) users needed to engage in at least 2 (or more) of the 8 lessons of a program, and (4) users should not have completed all 8 lessons of a program in a single day nor should the time period of use from the start date (either the comprehensive assessment or the first lesson) to the date of the last lesson have exceeded 6 months. Application of these criteria yielded a sample of 951 unique users.The following 5 criteria were established to select users appropriate to the study goals: (1) users had to be from a customer of per program varied between these groups with a decreasing number of lessons per any one program as the total number of programs used increased: Users of 1 program had an average of 3.44 lessons per program; users of 2 programs used had an average of 2.25 lessons per program; users of 3 programs used had an average of 1.80 lessons per program; and users of all 4 of the programs had an average of only 1.68 lessons per program. Note that at least one of the programs used by an individual who had used 2 or more programs, had to have a minimum of 2 lessons used.The final criterion was that if a student had used multiple iCBT programs during the 3-year period, then the experience data from only one program were included. Of the sample that met all 4 of the above inclusion criteria, most participants  used only 1 program, with the remaining 15.2% (145/951) having used multiple programs. Using more than 1 program was defined as using at least 1 lesson of 2 or more different programs during the study period. More specifically, 122 (12.8%) students had used 2 programs, 19 (1.9%) students had used 3 programs, and 4 (0.4%) students had used all 4 programs. The average number of lessons The choice of which program’s data to use for each student with multiprogram status was based on several criteria. Listed in order of importance, these criteria included: (1) clinical status on the symptom assessment at the start, (2) a higher number of lessons used (two minimum), (3) earlier start date in the study period, and (4) a longer time period for program use. For example, if a student had met the first set of criteria for use of 2 programs and had a score above the clinical score cutoff for the depression program but also a score that was below the clinical score cutoff for the insomnia program, then the data for the former was retained but not of the latter. To continue, if a student was above the cutoff scores for the severity symptom measures for both programs, then the program that had more lessons was retained. For example, if the depression program had 5 lessons and the insomnia program had 3 lessons, then the depression program was retained. Application of these criteria resulted in the following final mix of participants with data from only one program used: Of the 951 total unique users in the study, 347 (36.5%) users for the depression program, 325 (34.2%) users for the stress, anxiety, and worry program, 203 (21.3%) users for the social anxiety program, and 76 (8.0%) users for the insomnia program.Each of these measures of clinical symptom severity is a published, reliable, and validated scale from the scientific literature. Within each program, the symptom measure was repeated in every lesson. These 4 measures were aggregated in the comprehensive assessment .feeling nervous, anxious or on edge and (2) not being able to stop or control worrying. The instructions state: “Over the last 2 weeks, how often have you been bothered by any of the following problems?” Items are rated on a 0 to 3 scale. Ratings on the items were summed and scores were categorized into levels of severity: low 0-4, mild 5-9, moderate 10-14, and severe 15-21. The clinical status for anxiety was defined as moderate or higher (score of 10+). The severity mix for general anxiety in the sample was 52.6% moderate (171/325) and 47.4% severe (154/325).The generalized anxiety disorder 7-item scale was used to assess symptoms of anxiety . This isLittle interest or pleasure in doing things and feeling down, depressed, or hopeless. Items are rated on a 0-3 scale. Scores on the 9 items were summed and then categorized into levels of severity: minimal 0-4, mild 5-9, moderate 10-14, moderately severe 15-19, and severe 20-27. The clinical status for depression was defined as moderate or higher (score of 10+). The severity mix for depression in the sample was 32.3% moderate (112/347), 44.7% moderately severe (155/347), and 23.0% severe (80/347).The patient health questionnaire 9-item scale was used to assess symptoms of depression . This scThe social phobia inventory (SPIN) was used to assess symptoms of social anxiety . Past reget enough sleep to feel rested upon waking in the morning? (5) awaken short of breath or with a headache? (7) have trouble falling asleep? (8) awaken during your sleep time and have trouble falling asleep again? (9) have trouble staying awake during the day? and (12) get the amount of sleep you needed? The recall period was slightly modified for use by Learn to Live, such that in the preliminary comprehensive assessment, the instructions for this scale used the past 4 weeks reference time period, whereas in each lesson of the program, the instructions had a reference time period of the past week. The 6 items were rated on a scale of 1-6. The ratings were weighted  and summed. The total score was then categorized into 4 levels of severity: minimal 0-29, mild 30-43, moderate 44-60, and severe 61-100. The clinical status of insomnia was defined as moderate or higher (score of 44+). The severity mix of insomnia in the study sample was 55.3% moderate (42/76) and 44.7% severe (34/76).To assess symptoms of sleep disturbance and insomnia, the sleep scale from the medical outcomes study (MOS) developed by the Rand Corporation . The MOSThe data source for the score at the start of program use was most often the student's score from the preliminary comprehensive assessment, which had been completed by almost 9 out of every 10 participants . For those who did not complete the comprehensive assessment, the score for the start of program use was taken from the symptom assessment done as part of the first lesson. The data source for the last score at the end of program use was taken from the student's score on the last lesson used, which varied within person from lesson 2 to lesson 8. Each program had a full range of lessons represented from 2 to 8.Learn to Live services were sent an email and invited to complete a self-report survey about their experiences. Modest financial incentives were provided to students who participated in a follow-up survey. Note that offering incentives for survey completion was a routine component of business operations and not a procedure unique to the research study. The specific questions and response options and the findings are presented later in this paper. A total of 136 users completed a survey during the valid follow-up period, defined as at least 1 month but not more than 6 months after the date of the last use of the program (between 31 and 183 days after the date of the last lesson used). About 1 out of every 8 students in the study  completed a valid follow-up survey.All registered users of the Preliminary tests were performed by comparing the survey sample with the others in the total sample to determine the representativeness of the survey group. Specific statistical results for each of the measures compared are shown in All analyses were conducted using SPSS version 25 . DescripThis part of the paper describes the profile of college students based on demographic factors and their use of the 4 programs.gender diverse.The sample included 951 students from 4 colleges and universities, all located in the Midwest region of the United States. In the total sample, the average age of the users was 23.38 years (SD=6.54) and ranged from 15 to 62 years, although most users were in the 18 to 21 age group, typically of the college undergraduate experience. Females comprised the vast majority of program users at 74.2% (706/951), males were 23.6% (224/951), and 2.2% (21/951) of users self-identified as n=136). For race, 80.1% (109/136) identified as white, 8.1% (11/136) as Asian, 3.7% (5/136) as black or African American, 1.5% (2/136) as Hispanic or Latino, and another 6.6% (9/136) as Other or no answer. Most of the users who completed the survey were undergraduate students , although slightly more than a third were graduate students . Among the 85 undergraduates, the mix of school class was 15 freshman, 31 sophomores, 18 juniors, and 21 seniors. This profile indicates that these iCBT programs appealed to both undergraduate and graduate students who had a profile for age, gender, and race, which was consistent with the larger college student population in the United States [Other background factors of race and year in college were only collected in the follow-up survey  across the 4 programs. The average number of lessons used was 3.63 (SD=2.08) out of the 8 possible. The mean number of days of use from the start to the last lesson was 41.55 days (SD=43.77), with a median of 25 days and a range of 1 to 183 days. The period of time between the use of each lesson averaged 11.65 days (SD=13.14).This profile of program use is for the total sample . The number of lessons used was not related to gender  but was somewhat correlated with age , in that older students used more lessons. The duration of use was not related to gender  but was somewhat correlated with age , in that older students had a longer period of use.As expected, the number of lessons used and the duration of the period of use were strongly positively correlated . The use of a coach was strongly associated with participating in a greater number of lessons  and with greater duration of program use . In contrast, the use of a coach was only weakly associated with demographic factors of older age  and was unrelated to gender .About 1 in every 5 students chose to involve a teammate  for ongoing support . The use of a teammate was not associated with the number of lessons used , the duration of use , or age of the user . However, the use of a teammate was strongly associated with gender , such that females  were twice as likely to have used a teammate than were males .About 1 in every 8 students chose to engage a The demographic factors of age, gender, and operational use factors were also compared between the 4 iCBT programs. The results revealed significant differences between programs on 7 of the 9 factors tested .Both demographic characteristics of users differed significantly between programs. The average age of the students in the insomnia program was about 3 years older than the average age of the students in each of the other 3 programs. Although females were the majority of users in every program, the social anxiety program had relatively fewer women among users (64%) than the gender mix in the other 3 programs .The 4 programs were similar in the average number of lessons used per person. The percentage of users who completed a valid survey at follow-up after use was also similar across the 4 programs. However, significant differences between programs were present in 5 other utilization factors . The perThis part of the results examines the primary outcomes of the study for change in the clinical symptoms among users in each program separately. Changes from before to after program use in the level of severity of clinical symptoms were empirically examined in two ways. The first approach was more clinically focused and determined how many cases changed from being in clinical status at the start (100% of users by design) to no longer being in clinical status after use. The second approach compared the average levels of symptom severity across all cases in each program before and after use. Both approaches were performed separately for each of the 4 programs. See clinical status at the last lesson used was examined. The results showed that the percentage of users who changed from clinical status at the start to become lower and subclinical in their level of severity after use was 46% for stress, anxiety, and worry, 43% for insomnia, 36% for depression, and 22% for social anxiety. Averaged across the 4 programs (unweighted by different sample sizes), 37% of the students changed from clinical to non–clinical status after use.For each program, the number of cases that were still above the established scale score cutoffs for t test (two-tailed probability) and statistical effect size (Cohen d) analyses were performed for each of the 4 programs to test the extent of change in clinical symptom severity on average across all users from before to after use. The results indicated that each program had a significant improvement in severity. The average improvement (as a percentage of score reduction) for each program was as follows: 30% for stress, anxiety, and worry, 25% for depression, 22% for insomnia, and 16% for social anxiety. Each of these results had a statistical effect size that was considered as large (d range 0.80-1.25). Averaged across the 4 programs (unweighted by different sample sizes), there was a 23% reduction in the level of severity of clinical symptoms after use.Paired The findings in Part 1 revealed many differences between the 4 programs in terms of user characteristics and how the programs were used. Overall, there were also some correlations between many of the operational factors. This context indicated that the overall results found in Part 2 for clinical improvement specific to each program may be influenced by user demographics and operational factors. Given the different profiles of the 4 programs, it made sense then for us to explore changes over time in the clinical outcomes using other more sophisticated tests that take into account the joint influences of the user demographic and program use characteristics. This part of the results examined the impact of relevant potential moderator factors on the change in clinical outcomes in the full sample, considering all programs together.As each of the programs had different outcome measures and a standardized measure was needed for analyses involving all programs in the same tests in the total sample, we used the difference scores for the change in symptom severity measures for each program . These dA repeated measures analysis of variance model with covariates (ANCOVA) was conducted with the dependent measure of the difference score for the amount of change in symptom severity from start to last use. The factors included the program (4 groups), the covariates of the demographic factors of user age (number of years) and gender , the operational factors of use of the comprehensive assessment (yes/no), the number of lessons used (2-8), the time period of use (number of days), the use of a coach (yes/no), the use of a teammate (yes/no), and if the student had used more than one program (yes/no). Each continuous variable was used in the statistical tests as covariates with their full variance. The results for each factor are shown in the second column of n=534; mean=19.41 years, SD=1.11; median=19) and (2) those who were older and in the age range of 22 to 62 years .However, subgroups of the continuous variables were also created for descriptive purposes to better understand the results using the estimated scores. This recoding process was done for the user, the number of lessons, the user’s age, and the duration of use. The number of lessons used was recoded into 3 categories: (1) lessons used 2, (2) lessons used 3-7, or (3) all 8 lessons completed. The age of the student was recoded into 2 categories: (1) those who were within the traditional undergraduate age range of 21 years or younger , (2) middle time period , and (3) longest time period . Essentially, this yielded 3 groups that used the program for time periods that lasted approximately 1 week, 4 weeks, and 13 weeks.The time period of use was recoded into 3 categories, based on dividing the sample into thirds (which was done separately within each program): (1) shortest time period, (2) middle time period, or (3) longest time period. As expected, these 3 groups differed in the total number of days of program use: (1) shortest time period  by 3 factors. These 3 factors included the program topic, the number of lessons used, and the gender of the user.Overall, when adjusted for the program and all other factors, the typical college student user experienced a 24% reduction in the severity of clinical symptoms. This overall level of reduction in clinical outcomes was moderated to a significant degree , followed by the depression program (24%), the insomnia program (21%), and the social anxiety program (16%) as the lowest. Thus, the program effectiveness differed between the 4 programs by a range of 16% to 29%.A comparison of these adjusted results from the multivariate tests with the unadjusted results  indicateThe largest difference among the covariates was found for the operational factor of the number of lessons used. After controlling for which program was used and for all of the other operational and demographic factors, the students who completed the full program had an average improvement of 43%, compared with a 25% improvement among the students who used between 3 and 7 lessons, and only an 18% improvement for the students who used just 2 lessons. This was a relative difference such that the clinical success rate was 2.4 times higher for program completers than for those who participated minimally in doing only the first 2 lessons. This factor represents the level of dosage of the full content and intervention material. As this was the largest effect of all of the moderator factors tested, it was explored in more detail at each of the 7 specific levels of the number of lessons used .The general pattern of how the number of lessons used was associated with clinical change and distribution in the total sample is presented in F1,950=12.95; P<.001). Older students had slightly more clinical improvement after use than did the traditional college-age students .The gender of the user was significant as a covariate when controlling for all other factors . Males hP=.09). When tested combing the data from the only in the 2 programs with enough users of a teammate to provide a fair test , the students who had used a friend or family member for support had slightly more clinical improvement after use than the students who did not .The use of live support only approached significance as a moderator when controlling for other factors . StudentP<.001) when controlling for all other covariate factors. These results indicated that the users of both live supports had the most improvement (38%), followed by the users of one of the live supports (26%), and with the non-users of live supports users having the least amount of improvement (22%).However, when the 2 live support options were categorized into a combined variable of 3 groups , the findings were significant .Using more of the lessons on outcomes was critical for improvement. The lack of effect for the factor of multiple program user status makes sense when the average number of lessons In addition, both the comprehensive assessment and multi-program user factors had imbalanced distributions, with the vast majority of users being in 1 of the 2 categories . Nonequivalence of sample sizes between groups can also affect the fidelity of the comparison tests.Finally, additional repeated measures analysis of variance model tests with covariates were conducted to determine if the demographic and operational factors had moderator effects that were similar within each of the specific programs concerning their influence on the changes in the average level of symptom severity. The insomnia program, however, was excluded because it had too few users to allow us to conduct reliable tests of scores in the various subgroups of the factors. The analysis sample was also slightly smaller in each program tested, as the gender diverse students were excluded as they represented too small a group within each program for a reliable test .N=323), the results of the statistical analysis found that 2 of the 8 factors were significant as moderators of clinical symptom reduction after use , the results of the analysis found that 2 of the 8 factors were significant as moderators of clinical symptom reduction after use (P<.10). Males in the depression program had a slightly higher level of improvement than females . Older age students in the depression program had a slightly higher level of improvement than younger students . Other factors were not significant as moderators of improvement after use in the depression program.For the depression program (fter use . The effN=199), only one factor was significant in the statistical analysis as a moderator of clinical symptom reduction after use . For gender, males improved more than females in each program. The effect of age approached significance in the depression and social anxiety programs but not in the stress, anxiety, and worry program. The effect of the use of teammate was found to be significant for the depression program but not for the stress, anxiety, and worry program. Coaching contributed to slightly better outcomes, but was not significant for any of the 3 programs tested. Other operational factors did not affect the outcomes within each program.For the survey data, a descriptive approach guided the data analysis to explore user responses concerning the promotion of the services, experience with counseling, impact on college–related outcomes and attitudes, and overall user satisfaction with the service. The items and responses are presented in Other questions on the survey asked about the impact of the service use concerning 9 different academic related outcomes. The first set of questions examined positive academic behaviors. The results found that users were more likely after use to ask for help needed for classes (40%), to participate in class (29%), to complete assignments (15%), to make presentations in class (15%), and to go to class on time (12%). When combined, about 8 in every 10 students had one or more of these positive academic outcomes. A second theme involved how various adverse academic outcomes were reduced or avoided after use of the service. Some students reported that they were less likely to drop out of school (20%), were less likely to transfer away (15%), were less at risk of dropping a course (14%), or were able to avoid academic probation (10%). Considered together, about 1 in every 3 students who used the service had avoided experiencing at least one of these adverse outcomes. Overall, almost 9 in every 10 student users were successful in one or more of the full set of 9 academic outcomes.The results of another item indicated that about 8 out of every 10 students had a more favorable attitude toward the school because the school provided the web-based counseling service. Improved academic–related outcomes from the use of the program may have contributed to a better overall opinion about the school. Finally, a key finding of the survey was that almost 9 out of every 10 users were satisfied with their experience with the web-based program.getting more out of it since adding the web-based resources. Thus, the use of the iCBT tools had a positive effect on their ongoing therapy experience.The results of another item on the survey indicated that about half of the students , all of whom had met the clinical symptom threshold at the start of use, reported either a current or a past year experience with in-person counseling. Thus, about half of the students who used these digital mental health support tools already had experience with counseling from a live person. In addition, about a third of the users  were actively engaged in other in-person therapy at the time of their use of the iCBT program. This finding indicates that the use of the self-directed web-based support tool was adjunctive to in-person counseling for about 1 in every 3 of these students. In addition, among the subgroups of those who reported being currently involved with other therapies, half  reported that they were not use all of the lessons in a program. This was answered by 74 of the 136 survey respondents. Not having enough time to participate more often was the most common response for the subgroup of users . About 1 in 6 of these noncompleters indicated that they had improved enough to feel that they could stop before using more lessons . Another 1 in every 6 of these students did not complete the program because they were not able to relate to the content or did not find the program was helpful . Taken together, these comments suggest that using fewer than the full 8 lessons was influenced more by a variety of nonsystematic factors unique to the students involved than it was to the program content or functionality.The findings presented earlier on the importance of doing more of the lessons to achieve better outcome improvement were informed by responses on the survey for an item that asked why a student did This part of the results examines the replication of this study with another similar study of employee users of the same iCBT resources. The primary findings of this study are similar to those found in a recent study of the same 4 programs when used by employees . The samFor the demographic characteristics, the two samples were similar for gender  but, as expected, were quite different in age. The college sample users with an average age in the mid 20s were about 15 years younger than the average age of almost 40 years old for the employee user sample.The two samples were quite similar in terms of the operational factors , 36.8% of the college users had this outcome compared to 46.8% of the employee users.Next, the outcome of the average amount of change across all users in each sample was compared. The relative level of reduction in clinical symptoms for the typical user (as a percentage change from the mean score at the start to the mean score at the last use) was generally similar in each of the iCBT programs for the two samples. However, the employee users had a higher level of symptom improvement than the college users within each program. When averaged across the 4 programs (unweighted mean), the employee sample had an average of 28.9% reduction in symptom severity per person. This same metric was a 23.1% reduction for the college users. The results for clinical change by each program are presented in Perhaps the levels of the moderating factors of the number of lessons used and the use of coaching, which were both somewhat higher for employee users compared with college users, contributed to the slightly better clinical results for the employee user sample. Both these factors were also significant moderators of clinical improvement when tested in the study of employees involving both clinical and subclinical users . In summThis study examined how different technology-assisted support tools can reduce symptoms associated with anxiety, depression, social anxiety, and insomnia. All 4 of these programs had significant reductions in clinical symptom severity after use, and each program had results of a large statistical effect. When averaged across the programs and also controlling for relevant demographic and operational factors, the typical user had a 24% reduction in symptom severity. At the individual level, about 1 in every 3 users changed from being in clinical status at the start of use to being at a nonclinical level of severity after use. Our findings replicate the generally positive results found in other studies of college student users of behavioral health digital tools on clinical symptoms -21,25.dosage emerged as a particularly potent moderating variable, with much better results for those who participated fully in each program by completing all 8 lessons compared to those who participated minimally. Among program completers, the extent of improvement was more than double that of those who used only 2 lessons. This beneficial effect of adherence to the intervention (through completing more of the intended lessons) is a finding consistent with other research on adherence conducted in tests of similar kinds of iCBT tools for mental health issues [Therapeutic h issues ,39-41.Better clinical improvement among male users than among female users emerged as a surprise finding, even though less than a third of all users identified as male. One explanation could be the anonymity that this computerized medium affords and that there is no requirement to talk about one’s mental health challenges with another person. In this way, male users  may have felt less socially exposed by not having to talk with a counselor to better understand the nature of social anxiety. Male users may also be drawn more to the CBT oriented approach than to emotion-focused coping strategies, which are often emphasized in traditional talk therapy and which male college students tend not to prefer . Gender The use of optional live support was associated with an increased level of engagement in the key aspects of participation and yet was only weakly associated with better improvement in clinical symptoms, once these other more impactful factors were also considered in the same tests. In the total sample, however, the combination of using both live supports at the same time was significantly greater than using just one type of support or none at all. Coaching tended to be used more often and had a larger effect on the clinical outcome improvement than the options of live peer support from friends or family. The survey also asked participants to comment on their experience with using a coach. Two themes emerged from these qualitative comments as to why it was helpful. First, the coach provided accountability for engaging in more lessons and over a longer period of time. Second, the coach provided a caring, personalized form of support. The positive role of live coach supports found in this study has also been found in past research on technology tools for depression .Optional peer supports of a friend or family member as teammates were used far less often than coaching support . Note that coaches were also used more than teammates in the study of employee users of the same programs with the same pattern of very low use of teammates for the social anxiety and insomnia programs (see details in The survey data from the same users also revealed high levels of satisfaction and more favorable attitudes toward college or university. Program participation was also positively associated with at least one school performance outcome for 9 out of 10 students. About half of the students who used the web-based programs also had past year or concurrent experience in utilizing a face-to-face counselor. Moreover, one-fifth of the student users were referred to the digital services by the university counseling center, reflecting the acceptance of the digital services by the counseling centers and some existing integration of in-person and digital services. The benefits of using both traditional in-person and technological clinical supports have emerging research support .The results of this study replicate the findings obtained from a recent study of employee users of the same set of web-based programs from Learn to Live . The resAs in all applied research projects, there are certain limitations to this study. It was conducted using samples of college and university student users from multiple schools who had voluntarily used one particular commercial service. It is unknown whether these findings with this suite of tools can be replicated in other contexts of college student users. There were no comparison groups to assess the relative benefit from these iCBT tools compared to a matched group of nonusers. It is likely that some level of symptom reduction would occur naturally over time or form other causal forces not measured in this study. If so, whether the improvements over time in each program would have large size statistical effects if comparison groups of nonusers had been included in the study design is unknown. In addition, the causal mechanisms of how these web-based tools contribute to clinical improvements need further examination under more rigorous experimental study design conditions.This paper adds to the sophistication of the existing literature by comparing 4 distinct clinical topics, all of which shared the same digital platform and similar interactive website tools. Considered together, the variability in the number of lessons and in the total time period of use lends evidence to the flexibility that iCBT programs have to accommodate individual student preferences concerning when, how often, and how much to use the lessons. These iCBT tools appear to be associated with improved academic functioning and for some students, even the ability to stay in school. Additional study of outcomes beyond the typical focus on clinical symptoms in future studies would potentially add to the overall value proposition for web-based tools in support of the mental health of college students.