Translational Stroke Research (2020) 11:653–663 https://doi.org/10.1007/s12975-019-00747-2 ORIGINAL ARTICLE Association of High Serum Levels of Growth Factors with Good Outcome in Ischemic Stroke: a Multicenter Study Tomás Sobrino 1 & Manuel Rodríguez-Yáñez 1 & Francisco Campos 1 & Ramón Iglesias-Rey 1 & Mónica Millán 2 & Natalia Pérez de la Ossa 2 & Antonio Dávalos 2 & Raquel Delgado-Mederos 3 & Alejandro Martínez-Domeño 3 & Joan Martí-Fábregas 3 & Mar Castellanos 4,5 & Joaquín Serena 4 & Aida Lago 6 & Exuperio Díez-Tejedor 7 & José Castillo 1 Received: 1 August 2019 / Revised: 15 October 2019 / Accepted: 16 October 2019 / Published online: 25 November 2019 # The Author(s) 2019 Abstract The main objective of this research work was to study the association of serum levels of growth factors (GF) and SDF-1α with the functional outcome and reduction of lesion volume in ischemic stroke patients. In this multicenter study, 552 patients with non- lacunar stroke (male, 62.1%; mean age, 68.2 ± 11.4) were included within 24 h from symptom onset. The main outcome variable was good functional outcome (modified Rankin Scale [mRS] ≤ 2) at 12 months. Secondary outcome variable was infarct volume (in mL) after 6 ± 3 months. Serum levels of VEGF, Ang-1, G-CSF, BDNF, and SDF-1α were measured by ELISA at admission, 7 ± 1 days, at 3 ± 1 months, and 12 ± 3 months. Except for BDNF, all GF and SDF-1α serum levels showed a peak value at day 7 and remained elevated during the first 3 months (all p < 0.01). High serum levels at day 7 of VEGF (OR, 19.3), Ang-1 (OR, 14.7), G-CSF (OR, 9.6), and SDF-1α (OR, 28.5) were independently associated with good outcome at 12 months (all p < 0.0001). On the other hand, serum levels of VEGF (B, − 21.4), G-CSF (B, − 14.0), Ang-1 (B, − 13.3), and SDF-1α (B, − 44.6) measured at day 7 were independently associated with lesion volume at 6 months (p < 0.01). In summary, high serum levels of VEGF, Ang-1, G-CSF, and SDF-1α at day 7 and 3 months after ischemic stroke are associated with good functional outcome and smaller residual lesion at 1 year of follow-up. Keywords Growth factors . Infarct volume . Ischemic stroke . Prognosis Introduction [1]. Besides, the demographic change expected in Europe for the next 50 years suggests that this situation will become Improvement of the management of stroke patients, especially worse [2]. in developed countries, has contributed to the reduction in In addition, the ceiling of reperfusion treatment is already mortality and morbidity rates observed in recent decades. close (only 20% of with ischemic stroke patients can benefit However, the incidence of stroke has not followed this same from these treatments in highly specialized hospitals, whose trend showing a continuous increase in developed countries and their effectiveness does not exceed 50–60%) [3]. * Tomás Sobrino 3 Stroke Unit, Neurology Department, Hospital de la Santa Creu i Sant tomas.sobrino.moreiras@sergas.es Pau, Barcelona, Spain * José Castillo 4 Department of Neurology - Stroke Unit, Biomedical Research Jose.Castillo.Sanchez@sergas.es Institute of Girona, Hospital Universitario Doctor Josep Trueta, Girona, Spain 1 Clinical Neurosciences Research Laboratory, Health Research 5 Department of Neurology, Complexo Hospitalario Universitario da Institute of Santiago de Compostela (IDIS), Santiago de Coruña, A Coruña, Spain Compostela, Spain 6 Department of Neurology, Hospital Universitario La Fe, 2 Valencia, Spain Department of Neurosciences - Acute Stroke Unit, Hospital 7 Universitari Germans Trias i Pujol, Universidad Autònoma de Department of Neurology and Stroke Center, Neurosciences Area, Barcelona, Badalona, Spain IdiPAZ (Health Research Institute), La Paz University Hospital, Autónoma University of Madrid, Madrid, Spain 654 Transl. Stroke Res. (2020) 11:653–663 Moreover, new neuroprotective approaches are limited, main- stroke onset, consecutively admitted to Stroke Units of 6 ly due to a narrow therapeutic window. Therefore, the identi- Spanish hospitals. The period of recruitment of the patients fication of new targets for ischemic stroke is mandatory for was carried out in the first half of 2014. Patients without di- clinicians and researchers. agnostic confirmation by neuroimaging, lacunar infarcts, pre- The medium-long-term prognosis (weeks and/or months) vious disability (modified Rankin Scale > 1), cancer, severe of patients with ischemic stroke will depend on the size and systemic or metabolic disease, and inflammatory or infectious topography of the infarction, the speed and efficiency of the disease in the previous 15 days and patients who received acute phase therapeutic measures, and the degree of investigational drugs in clinical trials were excluded. neurorepair processes mediated by the patient’s cerebral plas- ticity mechanisms [4, 5]. Clinical Variables One of the main options for enhancing the mechanisms of cellular plasticity is cellular therapy that, favors by the extraor- All patients were admitted to an acute Stroke Unit and treated dinary advance of the investigation of cellular biology, repre- according to the European Stroke Organisation guidelines sents a hopeful therapeutic target for ischemic stroke. In this [15]. The follow-up period was 1 year, including 2 visits at 3 regard, the use of embryonic stem cells, induced pluripotent ± 1 months and at 12 ± 3 months. stem cells, and adult stem cells have demonstrated therapeutic Medical history recording potential vascular risk factors, effects in preclinical models of cerebral ischemia, and to a lesser blood and coagulation test, 12-lead electrocardiogram, chest extent in humans mainly through the mesenchymal stem cells. X-ray, and carotid ultrasonography were performed at admis- However, the mechanisms underlying cellular therapy are not sion. Stroke subtype was classified according to the TOAST well understood, since most of the transplanted cells disappear criteria [16], and stroke severity was assessed by a certified within a few weeks, and this therapy is not free from potentially neurologist using the National Institute of Health Stroke Scale serious complications [6–8]. It is possible that effectiveness of (NIHSS) at admission, 48 h, 3 ± 1, and 12 ± 3 months. cellular therapy is based on trophic factors and other bioactive Functional outcome was evaluated at 3 ± 1 and 12 ± 3 months substances that modify the cerebral microenvironment and using the modified Rankin Scale (mRS). Good functional out- stimulate the development of the patient’s own cellular niches come was defined as a mRS score ≤ 2. Physiotherapy/ [9–11]. Therefore, the stimulation of cerebral plasticity by using rehabilitation was collected during the hospitalization as well growth factors (GF), which participate in a coadjutant way in as the first 3 or 12 months of follow-up period. neurogenesis, neoangiogenesis, gliagenesis, and synaptogene- sis mechanisms, may constitute a more translational therapeutic approach from the lab to the bedside [12, 13]. Neuroimaging Variables Unlike the great existing knowledge on GF in preclinical models of cerebral ischemia, there is a gap in studies in clinical Cerebral computed tomography (CT) studies were carried out practice which may be one of the reasons why clinical trials at admission, between 4th and 7th days and after 3 months. with GF such as granulocyte colony-stimulating factor (G- Patients who received reperfusion treatment had another CT CSF) have failed [13, 14]. Thus, it is necessary to know better study before and at 24–36 h after treatment. Hemorrhagic the relationship between growth factors and the prognosis of transformation (HT) was assessed in the follow-up CT. HT ischemic stroke patients at long term. was defined as symptomatic when it was associated with early For this purpose, we designed a multicenter and observa- neurological deterioration (worsening > 4 points in the NIHSS tional study of a cohort of patients with acute ischemic stroke during the first 48 h from stroke onset). Lesion volume was in order to study the association of serum levels of GF (vas- calculated on the follow-up CT by using the formula 0.5 × A × cular endothelial growth factor [VEGF], [G-CSF], brain- B × C, where A and B are the largest perpendicular diameters, derived neurotrophic factor [BDNF], angiopoietin 1 [Ang- and C is the number of 1-cm thick sections that contain the 1]) and stromal-derived factor-1α [SDF-1α] with the func- lesion. All neuroimaging evaluations were made by investiga- tional outcome and reduction of lesion volume of ischemic tors who had no knowledge of the patients’ clinical and labo- stroke patients during a follow-up period of 1 year. ratory results. Outcome Variables Materials and Methods The primary endpoint was good functional outcome (mRS Study Population and Patient Characteristics score ≤ 2) at 12 ± 3 months; as secondary variables, we con- sidered the good functional outcome (mRS score ≤ 2) at 3 ± 1 We have conducted a multicenter and observational study of a months and the lesion volumes between 4th and 7th days and cohort of ischemic stroke patients, of less than 24 h from after 3 months. Transl. Stroke Res. (2020) 11:653–663 655 Laboratory Tests regression and multiple lineal regression models, respectively. The models were constructed considering the baseline vari- Biochemistry, hematology, and coagulation test were assessed ables related to each outcome variable. Results were expressed in the central laboratory of each participating hospital. as adjusted odds ratios (OR) or Beta estimate with their 95% However, the growth factors and SDF-1α determinations of confidence intervals (95% CI). The statistical analysis was the total patients included in each of 6 hospitals were per- conducted using IBM SPSS Statistic, version 20 (SPSS Inc. formed in the Clinical University Hospital of Santiago de Chicago, IL, USA). Compostela. The GF selected for this study were VEGF, G- CSF, BDNF, Ang-1, and SDF-1α. For these molecular deter- minations, venous blood samples were collected in Vacutainer tubes (Becton Dickinson, San Jose, CA, USA) at admission, 7 Results ± 1 days, 3 ± 1, and 12 ± 3 months. After allowing to clot for 60 min, blood samples were centrifuged at 3000×g for 10 min, Sample Description and the serum was immediately aliquoted, frozen, and stored at − 80 °C until analysis. Serum levels of VEGF, G-CSF, and A total of 650 patients with a first-ever ischemic stroke of less BDNF (Boster Biological Technology, Encyclopedia Circle than 24 h from symptoms onset was included in the study. Six Fremont, CA, USA), Ang-1 (Adipo Bioscience Inc., Santa patients were excluded due to lack of diagnostic confirmation, Clara, CA, USA), and SDF-1α (R&D Systems Inc., 39 patients were lacunar infarcts, 4 patients showed previous Minneapolis, MN, USA) were quantified using commercial disability (mRS > 1), and 12 patients were included in clinical ELISA kits following instructions provided by commercial trials during the acute phase. Moreover, 37 patients were loss houses. The coefficients of intra-assay variation were VEGF, during follow-up or suffered a recurrent stroke. Therefore, a final 4.4%; G-CSF, 5.2%; BDNF, 4.8%; Ang-1, 4.7%; and SDF- sample of 552 patients (male, 62.1%; mean age, 68.2 ± 11.4 1α, 3.6%; the coefficients of inter-assay variation were VEGF, years) was valid for testing the primary endpoint of the study. 6.9%; G-CSF, 8.3%; BDNF, 7.9%; Ang-1, 8.1%; and SDF- On the other hand, 28 patients died during the first month/ 1α, 7.2%. The determinations were performed in an indepen- week/admission, 37 at 3 months, and 84 at the end of the dent laboratory that did not have access to clinical or neuro- follow-up period of 12 months. Additionally, blood samples imaging data. were not obtained in 8 patients at 3 months, and 23 patients had not blood sample at 1 year of follow-up. So, a baseline Statistical Analysis serum sample was available in 552 patients, at 7 days in 524, According to unpublished own previous results, 42% (p1 = 0.42) of ischemic stroke patients have a mRS > 2 at discharge and do not show a significant improvement during a 1 year of follow-up period. It has been calculated that if a sample of 545 individuals (n = 545) is included, an accuracy of 4.12% (ω = 0.41) will be obtained by means of a normal 95% asymptotic confidence interval (γ = 0.95) bilateral (c = 2). For the calcu- lation of the sample size, EPIDAT version 4.2 (www.sergas. es/Saude-publica/EPIDAT) was used. Results were expressed as percentages for categorical var- iables and as mean (SD) or median and interquartile range for the continuous variables depending on their distribution. Kolmogorov-Smirnov test was used for testing normality. Chi-square or Fisher test was performed to study categorical variables. The continuous were studied with the Student’s t or the Mann-Whitney tests. Spearman’s or Pearson’s analyses were used to test bivariate correlations. ANOVA was used to analyze the relationship between stroke subtypes and growth factors. Mixed analysis of variance (MANOVA) was used to study the effect of time, and groups (good and poor outcome) by time (baseline to 12 months) interactions on GF and SDF- 1α levels. The influence of growth factors on functional out- come and the lesion volume was assessed by logistic Fig. 1 Screening and enrollment into the study 656 Transl. Stroke Res. (2020) 11:653–663 at 3 months in 507, and finally 445 patients had serum sample percentage of serum levels of GF during the first week were as at 12 months (Fig. 1). follows: VEGF, 77.4 ± 111.8% (p < 0.0001); G-CSF, 67.2 ± Regarding neuroimaging, CT at admission was performed 98.8% (p < 0.0001); BDNF, − 2.1 ± 44.3% (p = 0.349); Ang- in all; the second CT performed between 4th and 7th days was 1, 69.6 ± 120.6% (p < 0.0001); and SDF-1α, 29.6 ± 31.4% (p available in 537 patients, while the final CT was completed in < 0.0001). At 3 months, only an increase of VEGF (p < 409 patients after 3 months (average time, 6.3 ± 2.7 months 0.0001) and G-CSF (p < 0.0001) serum levels in relation to after admission). the basal concentration persisted; the concentrations of BDNF NIHSS at admission was 11 [6, 17]. A total of 198 patients (p = 0.312), Ang-1 (p = 0.632), and SDF-1α (p = 0.078) were (35.9%) received intravenous fibrinolytic treatment within the similar to baseline. By contrast, at 12 months, serum levels of first 4.5 h. Following TOATS criteria, 13.9% of patients were VEGF (p = 0.169) and G-CSF (p = 0.418) were similar to diagnosed as atherothrombotic stroke, 50.0% cardioembolic, those at baseline, but BDNF (p < 0.0001), Ang-1 (p = and 36.1% as undetermined origin. The median (quartiles) of 0.016), and SDF-1α (p < 0.0001) levels were lower. mRS at 3 months was 2 [1, 3] and 1 [0, 3] at 12 months. Primary Outcome: Good Functional Outcome at 12 Temporal Profile of GF and SDF-1α Levels Months Except for BDNF, all GF and SDF-1α serum levels showed a Table 1 shows the main characteristics of patients by functional peak value at day 7 (all p < 0.01) (Fig. 2). The increase in outcome groups. Three hundred and fifty-one (68.6%) patients Fig. 2 (a–e) Temporal profile of GF (VEGF, G-CSF, BDNF, Ang- 1) and SDF-1α levels at admis- sion, 7 ± 1 days, 3 ± 1 months, and 12 ± 3 months. All GF and SDF-1α serum levels showed a peak value at day 7 and remained elevated during the first 3 months (all p < 0.01), except for BDNF Transl. Stroke Res. (2020) 11:653–663 657 Table 1 Baseline clinical characteristics, vascular risk Good outcome n = 351 Poor outcome n = 201 p value factors, stroke subtype, biochemical parameters and Age, years 66.9 ± 11.6 69.6 ± 9.4 0.001 neuroimaging findings in patients Males, % 61.3 63.7 0.586 with good or poor outcome at 12 History of hypertension, % 58.4 71.6 0.002 ± 3 months History of diabetes, % 19.4 27.9 0.026 Smoking habit, % 26.8 13.4 < 0.0001 Alcohol consumption, % 15.4 9.5 0.051 History of hyperlipidemia, % 41.3 55.2 0.002 History of ischemic heart disease, % 15.4 16.9 0.631 History of atrial fibrillation, % 24.8 34.8 0.014 History of heart failure, % 10.3 9.5 0.883 Previous disability (mRS < 1) 0 (0, 0) 0 (0, 0) 0.448 Statins prior to stroke, % 31.3 21.1 0.008 Axillary temperature admission, °C 36.4 ± 0.5 36.6 ± 0.5 0.080 Glucose levels, mg/dL 127.6 ± 50.4 143.2 ± 43.5 < 0.0001 Leukocytes, × 103/mmc 9.3 ± 6.1 9.1 ± 2.4 0.100 Fibrinogen, mg/dL 404.7 ± 133.4 439.6 ± 160.1 0.003 NIHSS at admission 8 (5, 13) 18 (14, 20) < 0.0001 Fibrinolytic treatment, % 39.0 30.3 0.043 Infarct volume (4th to 7th day), mL 16.8 ± 29.8 98.2 ± 94.1 < 0.0001 Hemorrhagic transformation, % 13.4 16.9 0.263 Statins after stroke, % 70.9 62.2 0.038 TOAST 0.113 Atherothrombotic, % 11.7 17.9 Cardioembolic, % 48.1 53.2 Indeterminate, % 40.2 28.9 Rehabilitation during-hospital, % 27.1 33.0 0.145 Rehabilitation during 12 months, % 10.3 18.9 0.007 showed good functional outcome at 12 months. Patients with independently associated with good functional outcome at 12 good functional outcome were younger, showed milder stroke months (Table 2). severity, and smaller lesion volume at 4th–7th day. Furthermore, fibrinolytic treatment, rehabilitation, and statins Secondary Outcome: Good Functional Outcome at 3 treatment previous and during acute phase of stroke were more Months frequent in the group of patients with good outcome. On the other hand, with the exception of BDNF, all GF and Three hundred and forty-seven patients (62.9%) had good SDF-1α showed a different expression profile in patients with functional outcome at 3 months. Bivariate analysis showed good and poor outcome. Mean (SD) levels of VEGF, G-CSF, similar results to those obtained for the functional prognosis and SDF-α at day 7 and 3 months as well as Ang-1 at day 7 at 12 months (data not shown). The logistic regression analy- were significantly higher in patients with good functional out- sis showed that serum levels of SDF-1α (OR, 20.6; 95% CI come (Fig. 3). 9.2–46.1), VEGF (OR, 12.6; 95% CI 5.7–27.7), Ang-1 (OR, In the logistic regression analysis, serum levels of SDF-1α 9.1; 95% CI 4.0–20.8), and G-CSF (OR, 6.2; 95% CI 3.2– (OR, 28.5; 95% CI 13.2–81.2), VEGF (OR, 19.3; 95% CI 21.3) at day 7 were independently associated with good func- 10.7–231.9), Ang-1 (OR, 14.7; 95% CI 4.4–61.9), and G- tional outcome at 3 months. CSF (OR, 9.6, 95% CI 6.9–55.3) at day 7 were independently associated with good functional outcome at 12 months after Secondary Outcome: Lesion Volume at 4th–7th Days adjustment by significant variables of bivariate analysis and at 6 Months showed in Table 1. Moreover, serum levels of SDF-1α (OR, 20.2; 95% CI 8.1–57.1), VEGF (OR, 10.9; 95% CI 2.3–50.7), Figures 4 and 5 show the correlations between GF and SDF- and G-CSF (OR, 8.9; 95% CI 4.4–34.7) at 3 months were also 1α serum levels, measured at admission and the lesion 658 Transl. Stroke Res. (2020) 11:653–663 Fig. 3 (a–e) Temporal profile serum levels of GF (VEGF, G-CSF, BDNF, Ang-1) and SDF-1α in patients with good or poor outcome at 12 ± 3 months volumes at days 4–7, the correlation between GF and SDF-1α and NIHSS at admission), serum levels of VEGF (B, − 21.4), serum levels at day 7, and lesion volume at 6 months. Only G-CSF (B, − 14.0), Ang-1 (B, − 13.3), and SDF-1α (B, − admission serum levels of SDF-1α (r = − 0.373) were associ- 44.6) measured at day 7 were independently associated with ated with lesion volume at 4th–7th day. However, serum lesion volume at 6 months (all p < 0.01). By contrast, only levels at day 7 of VEGF (r = − 0.324), G-CSF (r = − 0.140), SDF-1α levels (B, − 29.6) at admission were independently Ang-1 (r = − 0.216), and SDF-1α (r = − 0.580), but not associated to lesion volume at 4th–7th day (Table 3). BDNF, had a significant correlation with the lesion volume after 3 months. In the multiple linear regression model, adjusted for those Discussion significant variables associated to lesion volume in the univar- iate analysis (history of arterial hypertension, alcohol con- This prospective and multicenter study shows a relationship sumption, history of atrial fibrillation, fibrinolytic treatment, between serum GF and SDF-1α and brain injury in patients Transl. Stroke Res. (2020) 11:653–663 659 Table 2 Adjusted and non- adjusted OR of good outcome at Growth factors Non-adjusted Adjusted* 12 months for serum levels of GF (VEGF, G-CSF, BDNF, Ang-1) OR 95% CI p OR 95% CI p value and SDF-1α at admission, day 7 ± 1, 3 ± 1 months, and 12 ± 3 VEGF at admission 1.7 1.3–2.3 0.001 1.7 1.5–4.9 0.001 months VEGF at 7 days 26.6 14.1–48.7 < 0.0001 19.3 10.7–231.9 < 0.0001 VEGF at 3 months 22.3 11.7–42.5 < 0.0001 10.9 2.3–50.7 < 0.0001 VEGF at 12 months 3.1 2.0–4.9 < 0.0001 1.6 1.1–10.0 < 0.0001 G-CSF at admission 0.8 0.5–1.1 0.255 0.85 0.4–1.5 0.603 G-CSF at 7 days 15.9 8.8–28.9 < 0.0001 9.59 6.9–55.3 < 0.0001 G-CSF at 3 months 6.5 3.7–11.4 < 0.0001 8.99 4.4–34.7 < 0.0001 G-CSF at 12 months 1.4 0.9–2.3 0.124 1.11 0.5–2.3 0.781 BDNF at admission 1.3 0.7–2.1 0.305 0.69 0.2–1.9 0.494 BDNF at 7 days 2.2 1.4–3.6 0.001 2.69 0.9–6.9 0.139 BDNF at 3 months 1.9 1.1–3.1 0.017 1.48 0.5–3.6 0.395 BDNF at 12 months 1.3 0.7–2.4 0.337 1.75 0.5–5.1 0.308 Ang-1 at admission 0.8 0.5–1.2 0.279 1.47 0.7–2.7 0.230 Ang-1 at 7 days 29.6 15.4–57.1 < 0.0001 14.72 4.4–61.9 < 0.0001 Ang-1 at 3 months 1.5 0.9–2.2 0.062 1.57 0.7–3.4 0.251 Ang-1 at 12 months 1.3 0.8–1.9 0.293 1.26 0.6–2.6 0.536 SDF-1α at admission 22.1 10.1–126.8 < 0.0001 16.25 10.1–87.2 < 0.0001 SDF-1α at 7 days 31.9 15.8–214.1 < 0.0001 28.53 13.2–81.1 < 0.0001 SDF-1α at 3 months 29.6 10.3–103.8 < 0.0001 20.19 8.1–57.2 < 0.0001 SDF-1α at 12 months 2.3 1.0–5.3 0.043 2.09 0.6–7.8 0.269 *Adjusted by age, history of hypertension, history of diabetes, smoking habit, history of hyperlipidemia, history of atrial fibrillation, statins prior to stroke, glucose levels, fibrinogen, NIHSS at admission, fibrinolytic treatment, infarc volume, rehabilitation during 12 months with ischemic stroke. The increase of serum levels of VEGF, outcome of ischemic stroke patients (in our series there was Ang-1, G-CSF, and SDF-1α at day 7 and 3 months after a slight non-significant association with a lower infarct vol- ischemic stroke was independently associated with a good ume); moreover, the increase of the BDNF expression after functional outcome at 3 and 12 months. This favorable effect rehabilitation has also been reported [19]. Other studies sup- on the primary variable was supported by positive effects on ported the lack of correlation between serum BDNF levels and the reduction of lesion volume after 3 months. Importantly, lesion size or recovery in stroke patients [20, 21]. However, it these effects remained significant after adjustment for well- is important to note that different research works have showed recognized prognostic factors such as age, lesion volume, that serum BDNF may not accurately reflect BDNF concen- stroke severity, and fibrinolytic treatment. trations in the brain due to endothelial dysfunction [22, 23]. Serum levels of VEGF, G-CSF, Ang-1, and SDF-1α BDNF levels could be also influenced by a variety of factors showed a similar biological pattern, with a peak value at day including platelet count, gender, smoking status, depression, 7 and remained elevated levels during the first 3 months. Of all and age and Val66Met polymorphism [21, 24–28]. Therefore, of them, SDF-1α was the biomarker more powerful associated further studies are necessary to elucidate the potential role of to the outcome variables (functional recovery and residual BDNF as a GF able to induce recovery in ischemic stroke lesion volume). Conversely, BDNF had a different profile, patients. showing a progressive decrease of its serum levels from the The selection of the GF analyzed in this study was per- stroke onset. In fact, BDNF levels were not associated with formed due to previous studies, mainly in preclinical any of the endpoints analyzed in this study, neither with de models of cerebral ischemia, since there is a gap in studies infarct volume, nor with the functional outcome during a in clinical practice which may be one of the reasons why follow-up period of 1 year. The role of BDNF in the patho- clinical trials with GF such as G-CSF have failed [13, 14]. physiology of ischemic stroke has been evaluated in animal Specifically, VEGF is a potent angiogenic factor, inducible and clinical studies, where the results were not conclusive. by hypoxia, which plays an important role in the vascular Previous studies [17, 18] have presented a relationship be- response to ischemia, promoting the formation of new ce- tween a baseline and isolated BDNF determination and rebral blood vessels (neovascularization) [29–34]. 660 Transl. Stroke Res. (2020) 11:653–663 Fig. 4 Scatterplots between GF (VEGF, G-CSF, BDNF, Ang-1) and SDF-1α serum levels mea- sured at admission and 7 ± 1 days and the lesion volumes at 4th–7th day Likewise, VEGF has been also implicated as a factor pro- morphogenesis, and plasticity of dendritic spines, resulting moting neurogenesis in the adult brain, increasing the pro- in synapses with functionality [40, 41]. liferation and differentiation of endothelial and neuronal The functionality of GF depends, to a large extent, on the progenitor cells [9, 29, 35]. relationship between them and between the cells that induce G-CSF induces the mobilization of hematopoietic stem their expression. Thus, VEGF induces the release of BDNF cells from the bone marrow to the injured brain regions, re- from endothelial cells [42] and stimulates the formation of ducing the volume of the infarct and improving neuronal plas- pericytes [31]. Similarly, Ang-1 and SDF-1α could be the link ticity and vascularization, which translates into a better func- between neurogenesis and angiogenesis, promote neuroblast tional recovery [36–38]. Also inhibits apoptosis and stimu- migration, and post-stroke functional recovery [43, 44]. lates the differentiation of neuronal progenitor cells, inducing The usefulness of the knowledge about the potential role of neurogenesis. GF in the recovery of the cerebral lesion caused by the ische- BDNF is a neurotropic factor involved in ischemia- mia is not conditioned only by a better understanding of the induced neurogenesis processes, and increased recruitment underlying mechanisms that mediate brain repair but also be- of endogenous progenitors to injured brain regions, medi- cause they may imply more translational therapies than those ating repair mechanisms and neuronal plasticity [39]. mediated by cellular therapy. In this regard, drugs that induce BDNF has also been shown to induce synaptogenesis, the expression of GF are already known, such as 4α-phorbol Transl. Stroke Res. (2020) 11:653–663 661 Fig. 5 Scatterplots between GF (VEGF, G-CSF, BDNF, Ang-1) and SDF-1α serum levels mea- sured at 7 ± 1 days and after 6 ± 3 months 12,13-didecanoate (an endothelial receptor agonist), 15(S)- clinical trials, although with modest results. The G-CSF has hydroxyeicosat acid (arachidonic acid metabolite) [34], or shown a slight functional improvement, but not of the neuro- guanosine [45]. In addition, GF have already been tested in logical deficit [14]. Table 3 Adjusted* B of lesion volume at 4th–7th day and after 3 Admission Lesion volume (4th–7th day) 7th day Infarct volume (at 6 month) months for serum levels of GF (VEGF, G-CSF, BDNF, Ang-1) B 95% CI p value B 95% CI p value and SDF-1α at admission and day 7±1 VEGF − 4.9 − 13.6 to 3.79 0.269 VEGF − 21.6 − 29.6 to − 3.7 < 0.0001 G-CSF − 1.4 − 11.1 to 21.42 0.125 G-CSF − 14.0 − 23.6 to − 4.7 0.003 BDNF 3.5 − 11.6 to 18.56 0.650 BDNF − 11.2 − 23.4 to 1.1 0.074 Ang-1 − 3.3 − 13.60 to 7.01 0.530 Ang-1 − 13.3 − 24.5 to − 2.0 0.021 SDF-1α − 29.2 − 41.95 to − 16.50 < 0.0001 SDF-1α − 44.6 − 54.2 to − 34.9 < 0.0001 *Adjusted by age, history of atrial fibrillation, NIHSS at admission, and, fibrinolytic treatment 662 Transl. Stroke Res. (2020) 11:653–663 This study has some limitations. First, patients were admit- Ethical Approval and Patient Consent This research was carried out in accordance with the Declaration of Helsinki of the World Medical ted to Stroke Units of 6 Spanish hospitals, and hematology Association and approved by the Ethics Committee of the participating and coagulation test were assessed in the central laboratory of hospitals. Informed consent was obtained from each patient or their rela- each participating hospital. However, patients were treated tives after full explanation of the procedures. according to the European Stroke Organisation guidelines [15]. In addition, the growth factors and SDF-1α determina- Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// tions of the total patients were performed only in one hospital. creativecommons.org/licenses/by/4.0/), which permits unrestricted use, Second, to have normal healthy subjects as a control group distribution, and reproduction in any medium, provided you give appro- (cohort of age, comorbidity) would give more value to the priate credit to the original author(s) and the source, provide a link to the results presented. Three, it is important to note that many Creative Commons license, and indicate if changes were made. conditions affect the level of growth factors, for example, subject exercise or age [21, 24–28]. Although given the mean age of patients (68.2 ± 11.4 years), their physical activity or exercise was low or moderate. 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