Low triglyceride to high-density lipoprotein cholesterol ratio predicts hemorrhagic transformation in large atherosclerotic infarction of acute ischemic stroke

The ratio of triglyceride (TG) to high-density lipoprotein cholesterol (HDL-C) is an objective approach to predicting poor outcomes in acute ischemic stroke (AIS). The impact of TG/HDL-C on hemorrhagic transformation (HT) after AIS remains unknown. The aim of this study was to explore the accurate effect of TG/HDL-C on HT after AIS. We enrolled a total of 1423 patients with AIS in the training cohort from a prospective, consecutive hospital-based stroke registry. Of the 1423 patients, HT occurred in 155 (10.89%) patients. The incidence of HT after AIS was significantly increased when there were low levels of TG (P=0.016) and TG/HDL-C (P=0.006) in patients with AIS attributable to large artery atherosclerosis (LAA), but not in those who suffered from cardioembolic stroke. After adjustment for covariates, a lower TG/HDL-C (OR=0.53, 95%CI=0.20-0.93) that was more than TG alone (OR=0.61, 95%CI=0.27-0.98) independently increased the risk of HT in LAA. Furthermore, our established nomogram indicated that lower TG/HDL-C was an indicator of HT. These findings were further validated in the test cohort of 558 patients with AIS attributable to LAA. In summary, a low level of TG/HDL-C is correlated with greater risk of HT after AIS attributable to LAA.


INTRODUCTION
Hemorrhagic transformation (HT) is a common complication of ischemic stroke in patients receiving recanalization therapy [1] that results in worse outcomes and delays the initiation of antiplatelet or anticoagulation therapy [2].HT results from damage to the blood-brain barrier and extravasation of blood over impaired cerebral vessels in acute ischemic stroke (AIS) [3,4].Previous investigators reported that the risk of HT and symptomatic intracranial hemorrhage was asso-ciated with old age, hypertension, reduced platelet count, large infarct, reperfusion time and thrombolytic treatment [5][6][7].However, the association between serum lipids and HT remains deeply controversial.
AGING triglyceride (TG) and TG/high-density lipoprotein cholesterol (HDL-C) are independently associated with mortality and poor outcome in AIS patients [13][14][15].Nevertheless, whether low levels of TG/HDL-C can increase the incidence of HT after AIS remains elusive.
The current study enrolled two independent cohorts of a total of 1981 AIS patients to explore the accurate effect of TG/HDL-C on HT after AIS, and we found that low levels of TG/HDL-C were correlated with HT in patients with AIS attributable to large artery atherosclerosis (LAA).Additionally, several nomograms have been successfully established to predict disease prognostics, but nomograms for assessing HT after AIS are scarce.Hence, we investigated the predictive power of a nomogram based on TG/HDL-C in AIS patients.

AGING
In patients with cardioembolism, HT correlated significantly with NIHSS score on admission (P=0.001) and with fasting glucose (P=0.001) as well as systolic blood pressure (SBP) (P=0.045),but not with lipid level (Table 2).However, baseline characteristics show-ed no correlation with the incidence of HT in patients with small-vessel occlusion (Table 2).For the test cohort, HT occurred in 50 (8.96%)patients.Similar results were observed in patients attributable to LAA (Table 3).

A novel predictive model
The univariate logistic regression analyses for baseline characteristics in the training cohort are shown in Table 4 and Table S1.The results showed that age, smoking, SBP, NIHSS scores, diabetes mellitus, coronary artery disease, atrial fibrillation, stroke etiology, prior use of antiplatelets, fasting glucose, platelets, TG, TC, LDL-C and TG/HDL-C were associated with HT (all P<0.05) (Table 4).The multivariable logistic regression analyses

AGING
showed that patients attributable to cardioembolism had higher risk of HT than those attributable to LAA (OR=3.03,95%CI=1.91-4.80,P<0.001); however, TG, TC, LDL-C and TG/HDL-C were not independently associated with HT in patients attributable to cardioembolism (P>0.05).To further investigate the association of stroke etiology with HT, we performed logistic regression analyses in patients attributable to LAA, cardio-embolism and small-vessel occlusion.The findings indicated that lower levels of TG (OR= 0.61, 95% CI=0.27-0.98,P=0.042) and TG/HDL-C (OR= 0.53, 95%CI=0.20-0.93,P=0.032) were significantly associated with a higher risk of HT in patients with LAA (Table 4), but not in patients with cardioembolism or small-vessel occlusion (Table S1).These results were further confirmed in the test cohort of patients with LAA (Table 4).

AGING
To investigate the predictive power of lipid level for HT in patients with LAA, we established a nomogram according to the results from univariable analyses to predict the incidence of HT.The univariable analyses showed that NIHSS scores on admission, diabetes mellitus, TG and TG/HDL-C were associated with HT in the training and test cohort patients with LAA.Furthermore, TG/HDL-C had the greatest AUC among lipid classifications, and NIHSS scores on admission, diabetes mellitus, and TG/HDL-C were included in the nomogram for both the training (Figure 1A) and test (Figure 1B) cohorts by stepwise logistic regression analyses.The novel models for both cohorts indicated that higher NIHSS scores, diabetes mellitus and lower TG/HDL-C were indicators of HT in patients with LAA.These findings were similar to those obtained previously in the multivariate logistic models.

Predictive values of TG, TG/HDL-C for HT
To study the predictive power of TG and TG/HDL-C, we performed ROC curves and AUC analyses regarding HT in patients attributable to LAA from the training cohort.We also analyzed the combined predictive value of TG and HDL-C for HT.The results showed that the predictive value of TG/HDL-C (AUC=0.77,95% CI=0.72-0.81)was significantly superior to TG (AUC=0.68,95%CI=0.63-0.72)and their combined effect (AUC=0.67, 95%CI=0.62-0.71)(Figure 2A).In addition, similar findings were observed when their predictive power was further analyzed by ROC curves and AUC analyses in patients attributable to LAA of the test cohort (Figure 2B).

DISCUSSION
Using the training cohort from a prospective consecutive hospital-based stroke registry in Nanjing First Hospital, we observed no independent correlation between lower LDL-C and HT but confirmed an association of lower TG/HDL-C with a high incidence of HT in patients attributable to LAA, but not cardioembolism and small-vessel occlusion.The significant association was further supported in the test cohort.The predictive power of TG/HDL-C for HT was superior to that of the other serum lipids.The nomograms further confirmed the potential significance of TG/HDL-C for predicting the incidence of HT in patients with LAA.
There has been a long-standing dispute regarding the increased risk of cerebral hemorrhage at lower levels of serum lipids.Several studies have reported that low cholesterol level was not associated with hemorrhagic stroke [18,19].Others have stated a positive correlation of cerebral hemorrhage and low cholesterol level [8].Lipid-lowering treatment with 80 mg atorvastatin has been shown to increase hemorrhagic stroke [20].

AGING
The underlying molecular mechanisms of the increase in cerebral hemorrhages at low levels of cholesterol remain unclear.Some investigators have suggested that adequate cholesterol levels may maintain the integrity of cerebral small vessels [21] and that immunoliposome may ameliorate endothelial damage and reduce HT after thrombolysis [22].Therefore, low levels of TG may increase the incidence of HT by injuring cerebral vessel integrity.
LAA is a central subtype of the TOAST classification system.A previous report found that, among patients with ischemic stroke, LAA was the cause for 12% to 54%, and cardioembolism was the cause for 10-26% [23].In the current study, LAA accounted for 50.74% in the training cohort.A major point distinguishing LAA from cardioembolism is stenosis of the proximal cerebral large artery, which may disturb relevant cerebral neurovascular units in various pathways.Furthermore, the microemboli increase the cerebral arterial stenosis, which results in a low perfusion state in the cerebrum [24,25].However, cardioembolism indicates heart-originating embolism without significant cerebral arterial stenosis [26].Hence, the cardioembolism could not induce long-standing subclinical damage for intact neurovascular units.Cerebral vessels of LAA patients, exposed to proximal arterial stenosis in the hemorrhage-prone state of low cholesterol, tend to increase the incidence of HT [17].
The TG/HDL-C was first reported to be associated with the presence of insulin resistance [28].Subsequently, TG/HDL-C predicted cardiovascular events in hypertension and diabetes mellitus [29,30].Our previous study indicated that low levels of TG/HDL-C were associated with mortality and worse short-term outcomes after AIS [14,15].This study has further shown that low levels of TG/HDL-C increased the risk of HT in patients attributable to LAA in two independent cohorts.Although there was a lower proportion of smoking in AIS patients with HT, as shown in Table 1, subgroup analyses by stroke etiology showed that smoking did not impact the incidence of HT in AIS patients of both cohorts.Additionally, the findings from nomograms by stepwise logistic regression analyses suggested that NIHSS on admission, diabetes mellitus and TG/HDL-C were also predictors of HT in the training and test cohorts, which further supported the results from multivariate logistic regression analyses.The predictive capacity of our nomogram was superior to that of NIHSS scores on admission.Therefore, TG/HDL-C should be considered in the prediction of HT in patients attributable to LAA.
A primary advantage of this study is that a relatively large number of AIS patients were enrolled from the Nanjing city; additionally, the training cohort was from a prospective consecutive hospital-based stroke registry in Nanjing First Hospital.Another advantage is the construction of a nomogram including TG/HDL-C, which accurately predicted the incidence of HT in patients with AIS attributable to LAA.Several limitations should be acknowledged regarding the interpretation of these results.For example, patients with AIS attributable to LAA were only enrolled in the test cohort, and these patients were not from a prospective consecutive hospital-based stroke registry.Additionally, we do not know whether dynamic changes of TG/HDL-C during treatment influence the incidence of HT in patients attributable to LAA.The underlying molecular mechanisms of low TG/HDL in HT should be deeply explored in further studies.
In conclusion, the present study suggests that low levels of TG/HDL-C may be related to an increased risk of HT in LAA, but not in cardioembolism.The negative association between TG/HDL-C and HT should be considered in the management of ischemic stroke attributable to LAA.

Study population and study protocol
The present study was approved by the Nanjing Medical University Ethics Committee and complied with the Declaration of Helsinki.All of the patient data were acquired from Nanjing First Hospital, Nanjing Medical University between January 2012 and June 2018 and from the Affiliated ZhongDa Hospital of Southeast University between January 2011 and January 2017.The patients were divided into two cohorts, including training and test cohorts.The clinical characteristics of the training and test cohorts are summarized in Tables 1  and 3. A total of 1423 AIS patients who were retrospectively collected from a prospective consecutive hospital-based stroke registry in Nanjing First Hospital were enrolled in the training cohort.An additional 558 AIS patients attributable to LAA who were admitted to the Affiliated ZhongDa Hospital of Southeast University were retrospectively recruited for the test cohort.Individual patients were enrolled in this study if they met the following conditions: (1) first ictus of stroke; (2) admission within 7 days of stroke onset; (3) blood sample extraction within 24 hours on admission (all enrolled patients had fasting lipid levels drawn the morning after hospital admission); (4) ischemic stroke; (5) had not received thrombolytic therapy with tPA and (6) standard in-house procedures were followed [15].Several patients were excluded as described in our previous study [15].

AGING
A multiset and hospital-based study was conducted to explore the precise effect of serum lipid levels on HT after ischemic stroke.Briefly, the association between serum lipid levels and HT was initially investigated in the training cohort.We found a significant association of serum lipid levels and HT in patients with AIS attributable to LAA by using a logistic regression model.Furthermore, we also constructed a prognostic model to predict the incidence of HT in AIS patients attributable to LAA.For the test cohort, we further confirmed the influence of serum lipid levels on HT in a total of 558 patients with AIS attributable to LAA, and we validated the prognostic model in another independent cohort.

Clinical and laboratory assessments
All patients underwent initial brain computed tomography (CT) or magnetic resonance imaging (MRI) before the initiation of antithrombotic therapy within 24 hours after admission.Subsequently, these patients underwent CT or MRI within 7 days after hospital admission or with any worsening.All CT or MRI scans were performed by a neuroradiologist blinded to the baseline characteristics and progress of AIS patients.The protocol of the MRI scan consisted of axial diffusion-weighted imaging (DWI), fluid-attenuated inversion recovery (FLAIR), and axial gradient echo (GRE) sequences.HT was defined as a high-density shadow within the area of low attenuation on the follow-up CT scan or a low-signal area within the acute ischemic lesion on the follow-up GRE sequences of the MRI scan.HT was confirmed according to the European Cooperative Acute Stroke Study I (ECASS I) [31].As symptomatic HT and asymptomatic HT are part of the same continuum and their discriminatory criteria are somewhat inaccurate, all HTs were considered independently of their clinical symptoms.Stroke etiology and stroke severity were determined as described in our prior study [32].

Figure 1 .
Figure 1.Nomograms of patients attributable to large artery atherosclerosis to predict hemorrhagic transformation after acute ischemic stroke.Locate the TG/HDL-C on the respective axis; draw a straight line up to the Points axis to determine how many points toward hemorrhagic transformation the patient receives for the TG/HDL-C; repeat this process for diabetes mellitus and NIHSS; add the points and locate this number on the Total points axis; and draw a straight line down to find the patient's estimated transformation.The c-indexes for the training and test cohorts of patients attributable to large artery atherosclerosis are 0.734 (A) and 0.698 (B), respectively.TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; NIHSS, National Institute of Health Stroke Scale.

Figure 2 .
Figure 2. Predictive power of TG, HDL-C, TG+HDL-C, and TG/HDL-C for hemorrhagic transformation in patients attributable to large artery atherosclerosis of the training (A) and test (B) cohorts.

Table 1 . Baseline characteristics of the training cohort according to the presence of hemorrhagic transformation.
AGINGBaseline characteristics of the training cohort according to stroke etiology are detailed in Table2.Data from an additional 558 patients attributable to LAA were retrospectively recruited into the test cohort.The median NIHSS score was 4. The baseline characteristics and mean concentrations of serum lipids are shown in Table

Table 1
For the training cohort, HT after AIS was observed in 155 (10.89%) patients.Comparisons of baseline characteristics in patients with or without HT are shown in Table1.Compared with patients without HT, patients with HT had higher proportions of no smoking, diabetes mellitus, coronary artery disease, atrial fibrillation and prior use of antiplatelets and anticoagulants; elder age; higher levels of SBP and fasting glucose; lower levels of platelets, TG, TC, LDL-C and TG/HDL-C; and higher scores of NIHSS (all P<0.05).The distributions of Trial of Org 10172 in Acute Stroke Treatment (TOAST) classifications in patients in the absence and presence of HT were significantly different (P<0.001) (

Table 2 . Baseline characteristics of the training cohort according to stroke etiology.
homocysteine; Lp-PLA2, lipoprotein-associated phospholipase A2; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG/HDL-C, TG to HDL-C ratio.Categorical variables were expressed as frequencies and percentages and were compared by the Chi-squared test.Continuous variables were expressed as the means ± standard deviations (S.D.) or medians (interquartile ranges, IQR), which were compared by the Student's t test, one-way ANOVA or the Mann-Whitney U-test if necessary.

Table 3 . Baseline characteristics of the test cohort according to the presence of hemorrhagic transformation.
protein; HCY, homocysteine; Lp-PLA2, lipoprotein-associated phospholipase A2.TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG/HDL-C, TG to HDL-C ratio.Categorical variables were expressed as frequencies and percentages and were compared by the Chi-squared test.Continuous variables were expressed as the means ± standard deviations (S.D.) or medians (interquartile ranges, IQR), which were compared by the Student's t test, one-way ANOVA or the Mann-Whitney U-test if necessary.

Table 4 . Univariate and multivariate analyses for the potential prognostic factors associated with hemorrhagic transformation by logistic regression model.
attack; INR, International Normalized Ratio; BUN, blood urea nitrogen; CRP, C-reactive protein; HCY, homocysteine; Lp-PLA2, lipoprotein-associated phospholipase A2; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG/HDL-C, TG to HDL-C ratio.a According to the modified TOAST classification.-, not available.a OR, adjusted for age, smoking, SBP, NIHSS on admission, diabetes mellitus, coronary artery disease, history of atrial fibrillation, stroke etiology, prior use of antiplatelets, fasting glucose and platelet; b OR, adjusted for SBP, NIHSS on admission and diabetes mellitus; c OR, age, SBP, NIHSS on admission and diabetes mellitus.