Prognostics signicance of small pulmonary vessels alteration measured by chest CT in connective tissue diseases with PAH

Pulmonary arterial hypertension (PAH) is characterized by structural alterations of the pulmonary vessels. Few studies have explored the clinical signicance of quantitative assessment of pulmonary small vessels by chest CT. Our aim was to assess whether the prognosis of connective tissue diseases (CTD)-PAH patients could be asscessed through pulmonary small vessels measured by chest CT. 42 CTD-PAH patients diagnosed based on right heart catheterization were retrospectively investigated. All patients underwent a chest CT within 1 month before and after RHC examination. Main pulmonary artery (MPA), the cross-sectional area of small pulmonary vessels < 5 mm 2 as a percentage of total lung area (%CSA < 5 ) were measured. The primary endpoint was a composite clinical worsening endpoint. hemodynamic parameters and quantitative CT parameters was assessed by Pearson’s correlation coecient. Receiver-operating characteristic curves were generated to assess the effectiveness of the %CSA <5 and MPA as targeting risk factors to predict the endpoint by evaluating the sensitivity and specicity of the scales. Results were expressed in terms of area under the curve (AUC) and 95% condence interval for this area. We dene the corresponding cutoff for each variable by the difference maximization method, and the patients were further divided into 3 groups according to the cutoff of the %CSA <5 and MPA. We use “pulmonary vasculature metrics” to dene classication based on the cutoff values of %CSA <5 and MPA. The prognostic value of selected baseline parameters was tested using Cox’s univariate proportional hazards regression analysis, and the variables that were signicant in the univariate model were then entered into a multivariate Cox model. The results were expressed as hazard ratios (HR) with 95% CI. Kaplan-Meier method was used to calculate time-to-event function among 3 groups and differences were assessed using the log-rank statistic. All P-values were two-sided and a P-value < 0.05 was considered statistically signicant.


Introduction
Pulmonary arterial hypertension (PAH), is a progressive disorder of the pulmonary circulation characterized by vascular remodeling within precapillary pulmonary arterioles leading to an increase in pulmonary vascular resistance (PVR) and right ventricular failure [1]. Connective tissue diseases (CTD) is one of the common causes of PAH, rank only to congenital heart disease and idiopathic pulmonary arterial hypertension (IPAH) in China [2]. The survival rate of CTD patients with PAH is much lower than that of patients without PAH [3,4], the prognosis of CTD-PAH and its response to targeted drugs are worse than IPAH [5][6][7].
Multi-parameters risk strati cation plays an important role in judging the prognosis of patients with PAH and guiding treatment decisions [1]. At present, the indicators in risk strati cation mainly come from the parameters of right heart catheterization (RHC), echocardiographic assessment of right heart function, and others such as 6-minute walking distance (6-MWD) and WHO function class. However, these indicators have a few limitations: RHC is an invasive diagnostic method and cannot be performed without an appropriate indication; besides, it is not feasible to be performed as a routine follow-up technique [8][9][10][11]. Echocardiography is limited by the operator experience and acoustic window, which substantially affect the accuracy and reproducibility of this approach [12,13]. The 6-MWD cannot be applied to patients with severe illness or patients who have di culty walking with lower limbs, and WHO function class is not absolutely objective [14].
As an objective and convenient method, chest CT can be used to evaluate the pulmonary parenchyma, pulmonary interstitial, and pulmonary vascular lesions, which is widely used in CTD patients [15]. A growing number of studies have shown that pulmonary vascular parameters measured on chest CT, including main pulmonary artery (MPA)diameter, the ratio of MPA to ascending aorta (MPA/AAO), have diagnostic and prognostic value in patients with PAH [16][17][18][19][20][21]. Our previous study showed that in patients with CTD-PAH, MPA greater than 37.7 mm was a potential independent risk factor of the poor long-term prognosis [22]. In addition, recent studies have shown that the cross-sectional area of small pulmonary vessels < 5 mm 2 as a percentage of total lung area (%CSA < 5 ) plays an important role in diagnosis and severity assessment of PAH and COPDrelated pulmonary hypertension [23][24][25][26][27]. However, the prognostic value of %CSA < 5 in PAH remains unknown. Therefore, here we will explore whether %CSA < 5 is associated with poor prognosis and can be used as a predictive indicator for screening high-risk populations of CTD-PAH patients.

Subjects
In the present study, we retrospectively analyzed the medical records and clinical characteristics of CTD patients diagnosed with PAH by RHC hospitalized in the First A liated Hospital of Nanjing Medical University between January 1, 2010 and September 1, 2020. Inclusion criteria were listed as follows: (1) a con rmed diagnosis of PAH according to the 2015 guidelines [1]; (2) meeting the diagnostic criteria for each subcategory of CTD: Systemic lupus erythematosus (SLE) was diagnosed according to 1997 American Rheumatism Association (ACR) criteria [28], Sjogren's syndrome (SS) was diagnosed according to 2002 international classi cation criteria [29], systemic sclerosis (SSc) was de ned according to 1980 ACR criteria [30]; mixed CTD (MCTD) was de ned by Sharp's criteria [31]; (3) having undergone chest CT within 1 month before or after RHC and the patients' condition was stable during the two examination intervals. The exclusion criteria included: (1) congenital heart disease; (2) other causes of precapillary PH (such as PH due to respiratory diseases, chronic thromboembolic PH, or other miscellaneous causes of PAH); (3) signi cant valvular heart disease of more than moderate to severe or LV ejection fraction < 50% diagnosed by echocardiography; (4) coexisting pulmonary conditions on computed tomography scan affected quantitative CT measurements: moderate or severe pulmonary interstitial brosis, current pneumonia, massive pleural effusion.
This study was approved by the Medical Ethics Committee of the First A liated Hospital of Nanjing Medical University (number: 2018-SR-333). As all variables were obtained retrospectively from available clinical data, the need for patients to sign informed consent was waived.
CT measurement of pulmonary vessels The diameter of the MPA and AAO was measured at the level of MPA bifurcation in its maximum dimension ( Figure 2A) [32].
To measure the CSA of small pulmonary vessels, three slices were selected. Three plain CT axial slices: 1 cm above the upper margin of the aortic arch (upper slice), 1 cm below the carina (middle slice), and 1cm below the right inferior pulmonary vein (lower slice).
Subsequently, images were analyzed with semiautomatic quantitative image-processing Image J software (version 1.48; National Institutes of Health, Bethesda, MD, USA). %CSA <5 we calculated according to the method reported by Matsuoka et al [23]. On each CT slice, %CSA <5 was obtained with the "Analyze Particles" function to count and measure objects on binary images, the number of vessels at a speci ed size and the CSA of each size range were obtained. Notably, the vessels that ran obliquely or parallel to the slice were excluded using the "Circularity" function in Image J ( Figure 2B-D) [24].

Clinical outcome
The primary endpoint was a composite clinical worsening endpoint (including all-cause mortality, worsening World Health Organization functional class, ≥15% reduction in 6-MWD, all-cause hospitalization, or the introduction of parenteral prostacyclin analog therapy) [33]. The time of follow-up was calculated as the time from RHC examination to the end of the study (September 1, 2020) or to the composite endpoint of clinical deterioration, whichever came rst. The follow-up data were obtained from hospital records. All patients were contacted to recon rm survival status by telephone personal interview of the patient or family members at the end of the study.

Statistical Analyses
All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) (ver 25.0, International Business Machines, Inc. Armonk, New York, USA). Qualitative data were expressed as frequency (percentage) and compared using χ 2 test or Fisher exact test. Quantitative data were expressed as mean ± standard or median (interquartile range [IQR]) and compared among groups by Student's-test or Mann-Whitney U test. The correlation between hemodynamic parameters and quantitative CT parameters was assessed by Pearson's correlation coe cient. Receiver-operating characteristic curves were generated to assess the effectiveness of the %CSA <5 and MPA as targeting risk factors to predict the endpoint by evaluating the sensitivity and speci city of the scales. Results were expressed in terms of area under the curve (AUC) and 95% con dence interval for this area. We de ne the corresponding cutoff for each variable by the difference maximization method, and the patients were further divided into 3 groups according to the cutoff of the %CSA <5 and MPA. We use "pulmonary vasculature metrics" to de ne classi cation based on the cutoff values of %CSA <5 and MPA. The prognostic value of selected baseline parameters was tested using Cox's univariate proportional hazards regression analysis, and the variables that were signi cant in the univariate model were then entered into a multivariate Cox model. The results were expressed as hazard ratios (HR) with 95% CI.
Kaplan-Meier method was used to calculate time-to-event function among 3 groups and differences were assessed using the log-rank statistic. All P-values were two-sided and a P-value < 0.05 was considered statistically signi cant.

Results
The baseline characteristics of CTD-PAH patients with or without endpoint events According to the inclusion and exclusion criteria, a total of 42 patients were enrolled retrospectively ( Figure 1).
Baseline demographics, clinical characteristics, hemodynamic, echocardiographic, and chest CT parameters are provided in Table 1. After a median follow-up time of 30.5 (IQR, 8.5-45.25) months, endpoint events occurred in 16 (38.1%) patients after 19.5 (IQR 10.0-45.5) months. As shown in Table 1, patients with endpoint events were characterized by elevated PVR, larger MPA diameter, and smaller %CSA <5 .

Relationships between hemodynamic parameters and quantitative CT parameters
The parameter related to small vessels, %CSA <5 correlated negatively with mPAP and PVR and positively with CI ( Figure 3). At the large vascular level, a positive correlation was found between mPAP and MPA/AAO Univariate Cox regression analysis was performed to identify variables affecting prognosis. We identi ed that hemodynamic parameter, PVR, and morphological CT parameters including MPA and %CSA <5 might be used as risk factors to predict the occurrence of endpoint events. Furthermore, we selected variables with a p-value<0.2, age and disease duration as candidate variables and entered into a stepwise multivariate Cox regression (Table 2) smaller MPA ( 36.75 mm). Kaplan-Meier analysis shown that the prognosis of group A was worse than that of group C (log-rank X 2 , 16.041; P=0.000) and group B (log-rank X 2 , 5.931; P=0.015), and that of group B was worse than that of group C (log-rank X 2 , 5.832; P=0.016) (Figure 4).

Discussion
In the current retrospective study, we found decreased small pulmonary vessels area (%CSA <5 ) and increased MPA diameter were associated with poor long-term outcome in CTD-PAH patients. The prognosis of patients with CTD-PAH satisfying %CSA <5 less than 0.382 and MPA greater than 36.75 mm is extremely poor.
We measured the pulmonary macrovascular diameter and %CSA <5 in CTD-PAH patients on chest CT. The mean diameter of MPA was 35.49 mm, which was signi cantly larger than the normal value of the previously reported in the Chinese population [34] and the MPA value of patients with endpoint events was higher than that of patients without endpoint events, %CSA <5 had a mean value of 0.79, similar to the previously reported values of healthy Chinese population [35], but the %CSA <5 of CTD-PAH patients with endpoint events was signi cantly lower than that in the rest of the patients. Our results showed that in CTD-PAH patients, %CSA <5 was negatively correlated with mPAP measured by RHC, consistent with previous studies. As early as 2009, Matsuoka et al found that in a population with severe emphysema and pulmonary hypertension, %CSA <5 was negatively correlated with mPAP measured by RHC [23]. Subsequently, a negative correlation between %CSA <5 and mPAP was also found in a study of 20 patients with PAH [27]. We speculate that the negative correlation between %CSA <5 and mPAP may be related to the following two factors: rst, impaired production of vasoactive mediators such as nitric oxide and prostacyclin in PAH patients, increased production of vasoconstrictors and proliferative mediators such as endothelin-1, ultimately lead to decreased vascular tone. This conjecture is supported by the signi cant negative correlation between %CSA <5 and PVR shown in Figure   3; Then, among the 42 patients with CTD-PAH included in our study, their mean PVR and mPAP were higher, and in these patients with severe PAH, right heart function is impaired to a certain extent, which may cause a reduction in cardiac output leading to distal atelectasis of small pulmonary vessels. This hypothesis was also supported by the positive correlation between %CSA <5 and CI shown in Figure 3, we can also see that the two echocardiographic parameters re ecting right ventricular function: TAPSE and FAC, were worse in patients with endpoint events than in those without an endpoint event, but unfortunately there was no statistical signi cance, possibly because of the small sample size of patients who underwent echocardiographic measurements of right ventricular function.
In this study, we documented that pulmonary vasculature metrics were associated with all-cause mortality and clinical worsening in patients with CTD-PAH in China. Several studies [22,[36][37][38] have shown that quantitative assessment of pulmonary large vessels can evaluate the prognosis of patients with PAH. Our results show that a combination of MPA diameter and %CSA < 5 could screen for patients with more severe disease and worse prognosis.
So far, few studies have used %CSA <5 to evaluate the prognosis of CTD-PAH. Shimizu et al [27]. reported the value of %CSA <5 in PAH patients was greater than that in the normal control group, but with the increase of mPAP, the value of %CSA <5 decreased. In studies of COPD-related PH [23,26,35], the value of %CSA <5 of COPD patients with severe PH was greater than that of COPD patients without severe PH, while mPAP was negatively correlated with %CSA <5 . In our study, %CSA <5 of CTD-PAH patients without an endpoint event were larger than the overall mean. This may be because of thickening of the intima/media/adventitia of the pulmonary arteries in CTD-PAH patients; on the one hand, this may be explained by elevated pressure within the pulmonary arteries, which likely expanded these small arteries before the vascular tone has not decreased to a certain extent, leading to larger %CSA <5 . Therefore, this result suggests that %CSA <5 may be more suitable as a prognostic indicator than a diagnostic indicator of PAH. Our ndings suggest that the quantitative evaluation of pulmonary small vessels by %CSA <5 combined with MPA has some value in improving risk strati cation in patients with CTD-PAH.

Limitations
There are several limitations to this study. First, this was a a single-centre retrospective study with a small sample size, there were also various deviations in the in-ow and discharge process. Second, Chest CT and RHC were not performed at the same time; however, we ensured that the interval between the two inspections did not exceed 1 month and during this period the patients' condition was stable. Third, since the speci city of the ROC analyses is not high for %CSA < 5 and MPA, the cutoff value of %CSA < 5 and MPA need to be further   Tables   Table 1 Clinical characteristics, hemodynamic parameters, echocardiographic RV parameters, and chest CT parameters of the overall population and patients with or without endpoint events.     Relationships between right heart catheterization parameters and %CSA<5. Abbreviations: mPAP, mean pulmonary arterial pressure; PVR, pulmonary vascular resistance; CI, cardiac index; %CSA<5, the cross-