Angiogenesis biomarkers discriminate multiple sclerosis phenotypes

doi:10.21203/rs.3.rs-4329965/v1

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Angiogenesis biomarkers discriminate multiple sclerosis phenotypes

https://doi.org/10.21203/rs.3.rs-4329965/v1

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Background: Multiple Sclerosis is a neuroinflammatory/neurodegenerative disease characterized by a state of “virtual hypoxia” in the central nervous system. Angiogenesis, one of the main homeostatic responses to hypoxia, has been implicated in the pathophysiology of multiple sclerosis; and angioneurins (angiogenic molecules released by/exerting effects on neural cells) are reported to have conflicting roles in perpetuating or ameliorating disease. This study aimed to determine whether angiogenic molecules are dysregulated in the serum and central nervous system of multiple sclerosis patients.

Methods: Serum samples were obtained from 317 multiple sclerosis participants (n=130 with relapsing-remitting multiple sclerosis; n=187 with progressive multiple sclerosis; n=43 controls) followed at the multiple sclerosis clinic in Calgary, Alberta, Canada. A proportion of participants were in trials of domperidone and hydroxychloroquine. Angiogenic factors were measured using the Human Angiogenesis Array & Growth Factor Array® multiplex (Eve Technologies). A meta-analysis of publicly available transcriptomic databases was performed to explore if the differences seen in serum were similar to those within the central nervous system.

Results: Several angioneurins were dysregulated in multiple sclerosis serum compared to healthy controls with increased expression of epidermal growth factor (p<0.01) and leptin (p<0.05). Further, multiple sclerosis phenotypes had distinct angiogenic signatures: epidermal growth factor was significantly higher in the sera of relapsing-remitting multiple sclerosis compared to progressive multiple sclerosis (p<0.0001), while endoglin was elevated in primary progressive (p<0.001) and secondary progressive (p<0.01) compared to relapse-remitting multiple sclerosis. Follistatin levels were exclusively higher in primary progressive compared to both relapse-remitting (p<0.001) and secondary progressive (p<0.0001) multiple sclerosis. Distinct angiogenic patterns were observed histologically in lesions and normal appearing brain tissue similar to what is seen in serum, with elevated epidermal growth factor across phenotypes, and elevated endoglin/follistatin in progressive multiple sclerosis lesions. Further, bone morphogenetic protein-9, endoglin, and follistatin were positively correlated with age and disability, while epidermal growth factor was negatively corresponded.

Conclusion: Angiogenesis is dysregulated in multiple sclerosis and across phenotypes. Angiogenesis may play complex roles in multiple sclerosis pathophysiology and be a relevant pathway, both in understanding disease mechanisms and as a possible therapeutic target.

progressive multiple sclerosis

RRMS

PPMS

SPMS

angiogenesis

angioneurin

hydroxychloroquine

domperidone

Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the CNS. Relapsing remitting multiple sclerosis (RRMS) is characterized by relapses and gadolinium enhancing lesions on MRI(1). Progressive MS (PMS) presents as steadily increasing unremitting disability either from disease onset (primary progressive MS: PPMS)(2) or with disability progression several years after an RRMS diagnosis (secondary progressive MS: SPMS)(1). Disease modifying therapies (DMTs) commonly used in the relapsing form(s) of MS have demonstrated only modest effects on disability accumulation, including in people with PMS that have continued inflammatory activity as manifested by enhancing lesions on MRI or recent relapses(3, 4).

Progression in the absence of inflammatory disease activity is far more difficult to treat, perhaps due to heterogenous mechanisms on a molecular and cellular level(5). One of several pathophysiological mechanisms suggested to have a prominent role in MS is a state of “virtual hypoxia” secondary to microglia/macrophage activation and the resultant oxidative stress.(5–7). One of the main homeostatic responses to hypoxia is angiogenesis(8), the formation of new blood vessels from pre-existing ones. An altered balance in angiogenic molecules has been shown to relate both to chronic inflammation and neurodegeneration(9, 10). Angiogenic molecules, such as vascular endothelial growth factor are increased in people with MS(9–12), whereas others, such as angiopoietin-2 and hepatocyte growth factor are decreased in people with SPMS who show disability worsening(13). Angiogenic molecules released by, and exerting effects on, neural cells have been termed “angioneurins”(14).

Uncontrolled angiogenesis increases vascular permeability and compromises the blood-brain-barrier (BBB) perpetuating inflammatory disease activity. Conversely, several angiogenic molecules have neurotrophic effects and can be protective within the CNS(15). Although angiogenesis is only one part of the multifaceted pathogenesis of MS, it has been postulated as a promising target for DMTs(9).

In this study, we hypothesized that the normally physiologically advantageous process of angiogenesis is dysregulated in MS, and that different markers would discriminate amongst phenotypes.

Study design and participants

Serum samples were obtained from 317 MS participants who were followed at the Calgary, Alberta, Canada MS Clinic. Forty-three individuals were recruited as healthy controls, matched for age and sex to RRMS/CIS patients, with no prior history of inflammatory, neurodegenerative, gastrointestinal disease, or treatment with DMTs. Blood samples were drawn from participants using a standard peripheral venipuncture by an experienced phlebotomist. Serum was isolated by centrifugation at 3000×g for 10 minutes, and samples were aliquoted and stored at − 80°C at the University of Calgary MS Biobank (a large repository of biomaterial for research purposes). MS study participants were categorized into two main phenotypes: RRMS and PMS (including both PPMS and SPMS). Participants with clinically isolated syndrome (CIS), or a first event of inflammatory demyelination, were grouped with the RRMS participants (n = 28/130).

In the RRMS cohort, treatment status with DMTs was recorded. DMTs were dichotomized into “high-efficacy” (fingolimod, B-cell depleting therapies, and natalizumab) and “lower-efficacy” (interferons, glatiramer acetate, teriflunomide, dimethylfumarate, and cladribine) groups. Recent inflammatory disease activity was defined as a clinical relapse diagnosed by an experienced neurologist, or new MRI T2 lesions (with or without new enhancing lesions) in the year prior to serum collection. Clinical disease severity and disability was rated according to the Kurtzke Expanded Disability Status Scale (EDSS)(16).

A proportion of the MS participants were enrolled in pilot or phase-2 trials conducted at the Calgary, Alberta, Canada MS Clinic; although all samples were processed and analyzed similarly (as described above). In the RRMS cohort, 16 participants were involved in a pilot trial of domperidone (Clinicaltrials.gov identifier NCT02493049). Participants in this trial were aged between 18 to 60 years, had a diagnosis of RRMS, were on a DMT, and had a gadolinium-enhancing lesion on clinically indicated screening brain MRI. Study participants had the gadolinium-enhanced brain MRI and serum samples drawn at baseline, 16 weeks, and 32 weeks. Baseline gadolinium enhancing lesions were followed over time with putative MRI measures of repair including Diffusion Tensor Imaging-derived Fractional Anisotropy (FA).

In the PMS cohort, 63 SPMS patients were involved in a phase-2 single-arm futility trial of Domperidone (Clinicaltrials.gov identifier NCT02308137)(17). Participants in this trial were aged between 18 to 60 years, had a diagnosis of SPMS, a baseline EDSS between 4.0 to 6.5, and a baseline timed 25-foot walk (T25FW) of ≥9 seconds. The individual data from this study has been described previously(13). Another sub-group of the PMS cohort included 39 PPMS patients who participated in a phase-2 futility trial of hydroxychloroquine (HCQ, Clinicaltrials.gov identifier NCT0291)(18). Participants in this trial were aged between 18 and 65 years, had a diagnosis of PPMS, and had no contrast-enhancing lesions on a screening MRI. Follow-up was 18 months and trial participants had longitudinal assessment of measures of disability including EDSS, T25FW, and nine-hole-peg (NHPT) tests. This population was divided into “responders” and “non-responders” based on if they experienced a ≥ 20% worsening on either the T25FW or the NHPT between baseline and 18 months of follow-up(19). Serum samples were drawn at baseline and 6 months.

Angiogenesis markers

The Human Angiogenesis Array & Growth Factor Array® multiplex (Eve Technologies, Calgary, Canada) was used to measure angiogenesis factors. Samples were run in duplicate. Proteins tested in this array included angiopoietin-2 (ANGPT2), bone morphogenetic protein-9 (BMP9), epidermal growth factor (EGF), endoglin, endothelin-1 (ETN-1), fibroblast growth factor-1 and 2 (FGF-1/2), follistatin, granulocyte-colony stimulating factor (G-CSF), heparin binding-epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin, placental growth factor (PLGF), vascular endothelial cell growth factor-A, C, and D (VEGF-A/C/D). Details of the multiplex method and limits of assay detection are available online at https://www.evetechnologies.com/product/human-angiogenesis-growth-factor-17-plex-discovery-assay-array-hdagp17/.

To investigate the expression of the selected biomarkers in the MS CNS, we performed a semi-quantitative meta-analysis of publicly existing transcriptomic databases. The keywords used for the search (performed on August 18, 2023) were “multiple sclerosis”, “brain”, and “spinal cord”, alone or in combination, in the Gene Expression Omnibus (GEO) database from the National Center for Biotechnology Information (www.ncbi.nlm.gov/geo/). Research type was limited to “humans”. The inclusion criteria for the dataset included (i) the dataset must be bulk mRNA-expression data supported by the literature; (ii) the data must be available for GEO2R analysis; (iii) each dataset must include ≥ 2 samples per group; and (iv) datasets would not include methylation/epigenetic analyses or focus on specific cell types. Disease phenotypes included per study were recorded unless unspecified in the original published study. Differentially expressed genes (DEGs) were defined through functional interpretation using GEO2R, and full DEGs lists were exported to an Excel Sheet (Microsoft Office) and then searched for the specific angiogenic molecules of interest.

Outcomes

The primary aim of this study was to test if angiogenesis markers were dysregulated in MS. To investigate this, serum angiogenesis markers were compared between MS participants and controls, as well as across MS phenotypes. Transcriptomic databases were interrogated to explore if there were similar differences in biomarker gene expression within the CNS in MS.

Secondary aims of this study explored the correlation between serum angiogenesis markers and demographic/clinical variables including disability, DMT use, age and sex. To investigate if angiogenesis markers were associated with potential remyelination, participants in the RRMS domperidone trial subgroup (Clinicaltrials.gov identifier NCT02493049, described above) were dichotomized into “good remyelinators” and “poor remyelinators” depending on the top and bottom 50 percentiles in FA change at 32 weeks. The sum of biomarker levels throughout the study period was then compared between groups. To investigate the longitudinal trajectory of serum angiogenesis markers and their association with HCQ treatment, the serum of participants in the PMS HCQ phase-2 trial subgroup (Clinicaltrials.gov identifier NCT0291, see above)(18) was utilized at baseline and 6 months.

Analysis and descriptive statistics

Baseline characteristics were summarized using frequency (percent) for categorical variables and mean ± standard deviation or median (interquartile range) for continuous variables. Normality of continuous variables was assessed by visual inspection (histograms) and use of the Shapiro-Wilk test. Groups were compared using the Student’s t-test, Mann–Whitney U test, one-way ANOVA, Kruskal-Wallis test, or Chi square test based on normality distribution using the Statistical Package for Social Sciences (SPSS 22.0, IL, USA) and GraphPad PRISM (PRISM 9.0, GraphPad Software, USA). A Bonferroni correction was used to adjust the multiplex analyses for multiple comparisons.

Pearson or Spearman correlation coefficients were computed to assess the relationship between continuous or ordinal variables. Values with results out-of-range (below the detectable limit) were transformed to zero; biomarkers with more than 33% of values outside of range were excluded from analysis in order to maintain the robustness, comparability, and generalizability of our findings(20). Statistical significance was taken to be at the two-tailed, 0.05 level. For analysis of longitudinal treatment effects, all available paired samples were included. Paired tests of significance were analyzed using the Wilcoxon Signed Rank test.

For the bioinformatic analysis of transcriptomic datasets we defined statistical significance based on adjusted p values of < 0.05 and recorded the fold-change associated with each gene. Comparisons amongst groups were made between normal appearing white or grey matter (NAWM or NAGM) and controls, and between white matter or grey matter lesions (WML or GML) and either controls (when available) or NAWM/NAGM. Given that different datasets were generated with different methods, including microarrays and/or Illumina platforms, we did not pool raw data for re-analysis, although raw data is publicly available in the GEO database.

Ethics

This study was approved by the University of Calgary conjoint Health Research Ethics Board (REB29-0900). All trial participants provided written informed consent before inclusion into the study.

Baseline characteristics and circulating angiogenesis markers

A total of 317 MS participants were included in this study where 130 (41%) had RRMS (n = 102 RRMS; n = 28 CIS), and 187 had PMS (n = 137 SPMS; n = 50 PPMS); matched to 43 healthy controls (mean age 42.1 ± 12.1 years). As expected, the PMS group was older (53.1 ± 7.3; p < 0.001 vs. other groups) and had higher EDSS scores (6.5, range 3.5–8.5; p < 0.001 vs. RRMS/CIS cohort) compared to the RRMS/CIS group (43.1 ± 9.9 mean age, 1.5 (range 0-6.5) EDSS). Sex was similar (% female) for controls (69.8%), RRMS/CIS (71.5%), and PMS (64.7%). Full baseline characteristics can be found in Supplementary Table 1.

At baseline, ETN-1, PLGF, FGF-1, FGF-2, G-CSF and IL-8 were undetectable in > 33% of all samples and were thus excluded from further analysis (see above in methods). In participants with MS, several of the angiogenic markers were positively correlated with each other while none displayed negative correspondence (Fig. 1A). BMP9, endoglin, and follistatin positively correlated with age, while EGF negatively associated with age (Fig. 1B).

Circulating angiogenesis markers are dysregulated across MS phenotypes and correlate with clinical disability

When analyzing angiogenesis markers at baseline, EGF (p < 0.01) and leptin (p < 0.05) were increased in RRMS patients compared to controls; there was a trend towards higher levels of HGF, VEGF-C, and VEGF-D in RRMS patients that did not reach significance (Fig. 2). None of the markers were meaningfully different between PMS patients and controls. However, across phenotypes, EGF was elevated in RRMS compared to PMS (p < 0.0001), while endoglin and follistatin were higher in PMS compared to RRMS (p < 0.0001 and p < 0.01 respectively, Fig. 2).

To further assess the distinct angiogenic signature across MS phenotypes, we separated PMS patients into PPMS and SPMS subgroups. EGF levels remained significantly higher in RRMS compared to both PPMS/SPMS subsets (p < 0.0001). Endoglin levels were elevated in PPMS (p < 0.0001) and SPMS (p < 0.001) compared to RRMS. Interestingly, follistatin levels were exclusively higher in PPMS patients compared to both RRMS (p < 0.0001) and SPMS (p < 0.0001). Finally, HB-EGF was higher in PPMS when compared to SPMS (p < 0.05) (Supplementary Fig. 1). No other angiogenic markers were different across phenotypes.

Finally, to delineate if angiogenic markers were associated with disability, baseline markers were compared to baseline EDSS scores. BMP9, endoglin, follistatin, and VEGF-A were positively correlated with EDSS, while EGF negatively correlated with EDSS (Fig. 3). Interestingly, this pattern (along with higher circulating levels of EGF in RRMS and endoglin/follistatin in PMS) was similar to the trends seen with age; wherein endoglin, and follistatin positively correlated and EGF was negatively associated with age (Fig. 1B). PMS patients tend to be older, and EDSS also tends to increase with age. To account for this all models were adjusted for age. After adjusting, baseline angiogenic levels remained the same and disability results were maintained for BMP9, EGF, and endoglin. There remained a trend towards correlation for follistatin which did not reach significance (p = 0.07). Table 1 summates relevant results from this study.

Table 1

Selected angiogenic markers are dysregulated in the serum and central nervous system in multiple sclerosis
Marker	Serum	CNS*	Age correlation	Disability correlation	Other
EGF	↑ in RRMS	↑ in NAWM/WML across all phenotypes	(-)	(-)
Endoglin	↑ in PMS	↑ in WML (PMS); ↓ in NAWM/WML (RRMS)	(+)	(+)
Follistatin	↑ in PPMS	↑ in NAWM (PMS)	(+)	(+)**	↑ in poor remyelinators
HB-EGF	↑ in PPMS	↑ in NAGM/GML (PMS), ↓ in WML (PMS)			↑ in active MS; ↓ with DMT
Leptin	↑ in RRMS	↓ in NAWM (RRMS), ↑ in GML (RRMS)
based on transcriptomic datasets (compared to MS normal appearing brain and controls). *Positive disability correlation with follistatin did not reach significance after adjusting for age (p = 0.07). DMT = disease modifying therapy; EGF = epidermal growth factor; GML = grey matter lesion; HB-EGF = heparin binding-epidermal growth factor; MS = multiple sclerosis; NAGM = normal appearing grey matter; NAWM = normal appearing white matter; PMS = progressive multiple sclerosis; PPMS = primary progressive multiple sclerosis; RRMS = relapsing-remitting multiple sclerosis; WML = white matter lesions

Circulating angiogenesis markers correlate with inflammatory activity

Of the 130 RRMS/CIS patients, 67 (51.5%) experienced inflammatory disease activity within 1-year before blood draw (defined as a clinical relapse or new T2 lesions on MRI). There were no sex differences between active and non-active groups. Active RRMS patients were younger (38.8 ± 9.3 years vs. 47.7 ± 8.3 years, p < 0.001) and had lower EDSS scores (1.5 (0.5) vs. 2.0 (1.5), p = 0.015). The only significant angiogenesis marker between groups was HB-EGF, which was higher in active RRMS compared to non-active RRMS (95.7 (61.1) pg/mL vs. 63.3 (35.3) pg/mL respectively, p < 0.01) (Table 1).

Eighty-one patients (62.3%) were treated with a DMT at the time of blood draw. The majority of stable RRMS participants were on a DMT, compared to active RRMS participants (n = 56/63 (88.9%) vs. n = 25/67 (37.3%) respectively, p < 0.001). Neither EDSS nor sex were different between treated and untreated groups, but DMT patients were significantly older (45.7 ± 9.1 years vs. 38.9 ± 9.6 years respectively, p = 0.001). Of the patients on DMTs, 50 (61.7%) were treated with a “lower efficacy” agent. There was no significant difference when stratifying angiogenesis markers according to DMT category (not shown). Intriguingly, HB-EGF levels were lower in people treated with a DMT (90.94 (57.1) pg/mL vs. 66.15 (47.3) pg/mL respectively, p < 0.01), the only significant difference found amongst biomarkers studied (Table 1). Using a linear regression model, HB-EGF levels were modestly associated with inflammatory disease activity independent of age/EDSS/DMT use (1.012 (95% CI 1.001–1.023), p = 0.037).

Transcriptomic changes are consistent with angiogenesis dysregulation in the MS CNS

To establish if angiogenic dysregulation exists in the CNS in MS, we performed a meta-analysis of published transcriptomic datasets. Our search yielded a total of 17 studies included in the meta-analysis, 16 including brain/choroid plexus samples and one with spinal cord samples (Supplementary Fig. 2). The selected datasets along with the associated study, population, and transcriptomic profiling method are listed in Supplementary Table 2.

All studied angiogenic biomarkers were identified in at least one dataset. In the MS normal appearing brain, a significant difference was observed in the expression of EGF, endoglin, follistatin, HB-EGF, HGF, leptin and VEGF-A in at least one dataset (Fig. 4). Generally speaking, leptin was decreased across datasets in the MS normal appearing brain compared to controls, while HGF was increased. Endoglin and VEGF-A were differentially expressed across datasets but meaningfully downregulated in at least one study; while EGF, follistatin and HB-EGF were differentially expressed across datasets but significantly upregulated in at least one study (Fig. 4). No observable differences were found in the expression of ANGPT2, BMP9, and VEGF-C/D.

Angiogenic differences in MS demyelinating lesions compared to MS normal appearing brain or controls

When focusing specifically on demyelinating lesions (compared to controls or MS normal appearing brain), a significant difference in the expression of EGF, endoglin, HB-EGF, HGF, leptin, and VEGF-A/C/D was found in at least one dataset (Fig. 5). EGF and HGF were primarily increased across datasets, and significantly upregulated in 2 or more studies. Endoglin, HB-EGF, leptin, and VEGF-A/C were differentially expressed across datasets, but meaningfully upregulated or downregulated in one or more studies. Only 2 studies identified VEGF-D, with one finding a downregulation in VEGF-D, and the other finding a non-significant upregulation (Fig. 5). No observable differences were found in the expression of AGPT2, BMP9 or follistatin.

Angiogenic differences across MS phenotypes and brain regions

Significant DEGs were then categorized according to disease phenotype/region described in each study (Supplementary Table 2). Several markers were dysregulated in all MS phenotypes. EGF was upregulated in at least one study in the NAWM/WML of all MS phenotypes. Similarly, HGF was upregulated in the NAWM and both WML/GML in MS. VEGF-A was upregulated in GML and downregulated in NAWM/WML.

Several angiogenic markers were only significantly changed in a specific MS phenotype: HB-EGF was downregulated in WML and upregulated in NAGM/GML, but only in PMS. Follistatin was exclusively upregulated in PMS NAWM. VEGF-C/D were only significantly different in PMS WML, with the former upregulated and the latter downregulated. Leptin was upregulated in GML and downregulated in NAWM, but only in RRMS. Finally, endoglin was downregulated in RRMS NAWM/WML but upregulated in PMS WML. Table 1 summates transcriptomic data for relevant angiogenic molecules.

Angioneurins are associated with remyelination and disability outcomes in clinical trial cohorts

A proportion of MS participants in this study were involved in pilot or phase-2 trials. In the RRMS cohort, sixteen patients were included in a pilot trial of domperidone, a potential remyelinating agent (Clinicaltrials.gov identifier NCT02493049, see above in methods). Longitudinal MRIs were obtained at baseline, week 16, and week 32 (Supplementary Fig. 3A). Percent change in lesion FA was used to group participants into “poor remyelinators” and “good remyelinators” based on if they were in the bottom or top 50th percentile change in FA at the above timepoints (Supplementary Fig. 3B). Angiogenesis markers were measured at the above timepoints, and follistatin was significantly higher in participants with poor remyelination (Supplementary Fig. 3C, Table 1). No significant differences were found with the other angiogenesis markers, or with demographic variables between these two groups (not shown).

Another subset of PPMS participants (n = 39) participated in a phase-2 HCQ futility trial (Clinicaltrials.gov identifier NCT0291)(18). As described above in methods, this population was divided into responders (those with no disability worsening) and non-responders (those with disability worsening) based on if they experienced a ≥ 20% worsening on the T25FWT or the NHPT between baseline and 18 months of follow-up(19). Baseline age, sex, and levels of disability (NHPT, T25FW, EDSS) were not observably different between responders and non-responders. Baseline follistatin levels were significantly higher in responders compared to non-responders (not shown); no other baseline angiogenic marker was different between the 2 groups.

After 6 months of hydroxychloroquine treatment, most of the angiogenic markers remained stable (not shown). Interestingly, ANGPT2 (p < 0.01), endoglin (p < 0.0001), and leptin (p < 0.001) were all increased, while VEGF-A (p < 0.01) decreased during this period (Fig. 6A). When separating the aforementioned angiogenic markers into HCQ responders and non-responders, there were significant changes in the responder group exclusively: with a decrease in follistatin and an increase in endoglin and leptin levels (Fig. 6B).

Angiogenesis markers are dysregulated in the serum/CNS in MS, and can discriminate phenotypes

In our study, we found that several angiogenic factors were dysregulated in MS which is consistent with existing literature, and that MS phenotypes have distinct angiogenic signatures, which has not been described previously.

Epidermal growth factor (EGF)

EGF expression was increased across all MS phenotypes (with significant sera elevation in RRMS compared to PMS and controls). When looking specifically at MS lesions two different datasets showed agreement of EGF overexpression in the NAWM and WML. Previous reports have demonstrated elevated levels of circulating EGF in MS(21), and higher levels of plasma EGF in RRMS compared to PMS(22). EGF is an angioneurin crucial for neuronal and glial cell lineage proliferation; in vivo EGF can be mobilized to demyelinated CNS areas and may be crucial in remyelination(23). Taken together, our preliminary data suggests an upregulation of EGF during active inflammation, and downregulation in the progressive forms of disease where there is less inflammation and remyelination failure is evident.

Endoglin

Endoglin is a transmembrane glycoprotein belonging to the transforming growth factor (TGF)-β superfamily (similarly to follistatin); it functions as an antagonist to activin-A and bone morphogenetic proteins. Endothelial endoglin expression is increased in chronic human MS lesions(24), and at least 2 reports have shown elevated circulating endoglin in RRMS(25, 26). However, this is the first study to find increased serum endoglin exclusively in PMS compared to RRMS and controls. In the CNS, endoglin was increased in WML in PMS, and reduced in NAWM/WML in RRMS. Endoglin has been associated with increased macrophage/microglial activation and neuroinflammation(27), an important mechanism in MS disease progression(5). An upregulation of endoglin in progressive MS may reflect pathogenic processes, or endogenous repair (as endoglin deficiency is detrimental to stroke recovery in mice)(28, 29).

Follistatin

Similarly to endoglin, follistatin is a transmembrane glycoprotein belonging to the TGF-β superfamily. In our study, follistatin was exclusively higher in the sera of PPMS participants; in the CNS it was significantly upregulated in PMS NAWM. A previous report found decreased monocyte production of follistatin in RRMS(30), but this is the first study to show increased levels of follistatin in PMS serum and brain. Although follistatin has been proposed as a negative regulator of neuroinflammatory responses(31), it also inhibits remyelination through antagonism of activin-A, an essential promoter of oligodendrocyte proliferation(32, 33). Herein, we found higher follistatin levels in poor remyelinators with RRMS, and higher levels in the PPMS population, where remyelination failure is thought to be a relevant mechanism driving progression(5).

Hepatocyte growth factor (HGF)

Plasma HGF is elevated in MS(11) and possibly higher in PMS compared to RRMS(22). This is consistent with our findings where HGF was increased in MS normal appearing brain compared to controls, and upregulated in NAWM, WML, and GML across all MS phenotypes. While this may suggest correlation with pathology, HGF has also been described as an angioneurin essential for functional recovery and remyelination. It promotes endogenous repair in spinal cord injury(34), lower levels are associated with disability worsening in SPMS(13), and exogenously supplied HGF promotes recovery in animal models of MS(35). Taken together this may suggest that higher levels of HGF in MS may actually be a reflection of endogenous repair processes stimulated by the natural progression of the disease, rather than a pathologic mechanism.

Heparin binding-epidermal growth factor (HB-EGF)

HB-EGF is a trophic factor implicated in neuronal survival and glial cell proliferation(36). It is thought to play a role in lesion formation; this is demonstrated by higher concentrations in activated astrocytes in both active and chronic active MS lesions(36). In addition, HB-EGF increases BBB permeability(37), an early feature in demyelinating lesion formation. This suggests a potential role for HB-EGF in astrocyte-mediated regulation of the BBB. In our study, circulating HB-EGF was higher in active RRMS, and lower in patients treated with DMT. Additionally, this is the first study to identify HB-EGF overexpression in the sera of PPMS patients compared to SPMS patients, and in PMS GML; we postulate that HB-EGF is involved not only in active inflammation, but also in potentiating the neurodegenerative process.

Leptin

Leptin is an adipokine strongly associated with obesity and metabolism. Previous studies of circulating leptin levels in MS are conflicting, with either elevation or no difference in leptin levels between MS and controls(38–41). In our study, we found elevated leptin levels in RRMS; these results may be confounded by body mass index, which unfortunately was not available as a variable for most participants. Further, leptin expression was decreased in the MS normal appearing brain compared to controls, overexpressed in GML, and underexpressed in WML/NAWM in RRMS. Leptin concentrations have been found to correlate with disability(39), the systemic pro-inflammatory response(38, 42), and MS risk(43). It may also have a role in promoting autoreactive T-cell proliferation and inhibiting T-regulatory cell proliferation to exacerbate the inflammatory response(38); this could explain its higher concentrations in lesion areas in active forms of MS (RRMS) compared to PMS.

Angiogenesis markers have relevance for resilience against injury and the effect of age

Dysregulation of angiogenic factors is present in the MS CNS as demonstrated in this study. Several authors have suggested that angiogenesis may exacerbate inflammatory injury(9, 44, 45), and anti-angiogenesis agents ameliorate disease activity in animal models of MS (9, 45–48). In this study, circulating HB-EGF was higher in active RRMS, while follistatin correlated with poor remyelination. Additionally, we found that several angiogenic molecules were associated with disability and age.

Baseline BMP9, endoglin, and follistatin levels correlated positively with EDSS, while EGF correlated negatively. Even after correcting for age, this result was maintained for BMP9, EGF, and endoglin. Although BMP9 positively correlated with disability (and is increased in neuromyelitis optica, a related neuroinflammatory disorder of the CNS)(49), our serum analysis did not reveal significant differences and we found no previous reports of serum BMP9 in MS. The role of BMP9 in MS remains to be elucidated. Interestingly, follistatin levels increased as disability increased, while EGF decreased. As described above, follistatin may inhibit remyelination(32, 33) while EGF is an angioneurin with pro-remyelinating properties(23). We speculate whether a loss of EGF in combination with increased follistatin could contribute to remyelination-failure in PMS.

Similarly to EDSS, BMP9, endoglin, and follistatin positively correlated with age, while EGF negatively correlated. There is likely a complex interplay of age, disability, and progression in MS, with age as the most important risk factor for disease progression(50). Ageing is a well-known factor determining the proper regulation of angiogenesis(51), and we postulate that age-related angiogenesis dysregulation in response to virtual hypoxia(6) may be a mechanism in MS neurodegeneration.

Angiogenesis markers play complex roles in CNS injury

The above interpretations are complicated by the varied roles that angiogenic factors likely play in neurodegeneration and neuroinflammation. Angiogenesis is generally quiescent in healthy adults with the exception of strictly controlled physiologic situations such as female reproduction, and tissue repair(52). Angiogenesis is an adaptive response to hypoxia, allowing increased oxygen and nutrients into areas of increased cellular needs. This advantageous process can be disrupted in diseases where chronic inflammation and a hypoxic environment are present, leading to over-activation of angiogenic factors(52). MS is one such disease fueled by chronic inflammation resulting in oxidative stress and mitochondrial dysfunction, neurodegeneration, and the creation of a virtually hypoxic CNS environment (Fig. 7)(5, 7).

Uncontrolled angiogenesis likely serves to increase vascular permeability and compromise the BBB, allowing an influx of inflammatory cells, nutrients, and oxygen into the CNS to perpetuate disease activity; it may also directly induce inflammation and alter the extracellular matrix (Fig. 7)(9, 44, 45). For example, endoglin activates microglia(27), leptin increases T-cell proliferation(38), and HB-EGF increases vascular permeability(37).

Conversely, angiogenic markers may also play a protective role in both vascular and neuronal cells (Fig. 7). Angiogenic molecules released by and exerting effects on neural cells have been termed “angioneurins”(14). For example, VEGF-A has both remyelinating and neuroprotective properties(53, 54), and may act as a neuroprotective agent in the late phases of MS(9). ANGPT2 plays a role in acute inflammation in animal models of MS, but increases in late stages of MS and improves CNS injury repair(55–57). EGF and HB-EGF are well known to promote oligodendrocyte differentiation and remyelination(23, 58), while HGF has neuroprotective and immune regulating properties in the CNS(59). BMP9 is essential for neurogenesis during development(60), and leptin can regulate microglial activity and promote remyelination by modifying oligodendrocyte signaling(61).

The complex and varied roles of angiogenic molecules in the CNS highlights the interplay of physiologically advantageous mechanisms such as angiogenesis, in a highly dysregulated system such as MS. It is important to recognize that because of this duality, studying individual angiogenic molecules will likely not to elucidate precise mechanisms in MS pathophysiology. Instead, several molecules should be explored simultaneously, as well as their complex interaction with their cellular sources (glia, monocytes, and endothelial cells).

Hydroxychloroquine (HCQ) may modify angiogenic factors in PPMS

Herein we found that HCQ treatment was associated with a significant increase in certain angiogenic molecules: ANGPT2, endoglin, and leptin, while VEGF-A was significantly reduced. In vitro HCQ directly reduces angiogenesis in cultured endothelial cells(62) and inhibits the production of ETN-1 in endothelial cells exposed to eclamptic sera(63). HCQ’s effect on VEGF-A remains uncertain; in one study HCQ reduced its expression in endothelial cells(64), however, another small study did not show any changes in patients with antiphospholipid syndrome(65). Furthermore, HCQ improves insulin sensitivity and glucose disposition in skeletal muscle(66), which may account for its increase of the adipokine leptin in our study. HCQ may also have indirect effects on angiogenesis by decreasing the activation of immune cells such as microglia(8, 67, 68), a predominant cell type in the pathophysiology of progressive MS and the creation of a hypoxic environment(5).

In our study, follistatin was higher at baseline in PPMS responders treated with HCQ compared to non-responders, which is congruent with the potential negative effects of follistatin in remyelination. Interestingly, when comparing the significant changes in angiogenic markers by month 6 on HCQ, only treatment responders had significant angiogenic changes in follistatin, endoglin and leptin. In particular, follistatin was significantly reduced after HCQ treatment in responders; this preliminary data suggests that changes in angiogenic molecules, as opposed to baseline levels, may be more reflective of HCQ´s biological/therapeutic effect. The clinical significance of such changes remains to be elucidated and warrants further study.

Limitations

A limitation of this study was that several of the MS individuals participated in different sub-group studies with variable DMTs, whose effect on angiogenesis remains unknown. Regardless of their original trial, participants were all followed by the same neurologists at the MS clinic in Calgary, Alberta, Canada, and had the same sample acquisition and storage. Additionally, serum samples in the RRMS/CIS group were matched by age to healthy controls; however, the same was not the case for the PMS population. We included age as a co-variable in regression analyses, but age is likely an important factor to consider independently. Another limitation of our study is in its primarily transversal design, where only a small portion of participants had longitudinal samples. This did not allow us to draw conclusions on the longitudinal trajectory of the biomarkers investigated. Additionally, angiogenesis marker analysis was done in serum, and it is unclear if this is reflective of what may be occurring in the CNS/CSF. Our analysis of transcriptomic datasets partially bridges this gap, but studies in matched serum/CSF samples are warranted.

In conclusion, angioneurins may play complex roles in MS neuroinflammation, neurodegeneration and repair. Herein, we found that several angiogenic factors were dysregulated in MS serum and CNS, and MS phenotypes had distinct angiogenic signatures. Further, specific markers such as follistatin and EGF correlated with clinically relevant features in MS including disability and age. Finally, angiogenic factors may reflect the biological/therapeutic effect of hydroxychloroquine in MS. The precise cellular origins of angiogenesis markers in the MS CNS has not been established and this study was not designed to draw mechanistic conclusions; more studies on the role of angiogenesis in MS are warranted.

Ethics approval and consent to participate

This study was approved by the University of Calgary conjoint Health Research Ethics Board (REB29-0900). All trial participants provided written informed consent before inclusion into the study.

Competing interests

H.Y, C.S., N.B., and Y.Z. have no relevant financial interests to disclose. C.L. received consult fees from EMD Serono, Novartis, Horizon Therapeutics, and Sanofi outside of this work. M.K. received consulting fees and travel support from Biogen, EMD Serono, Novartis, Roche, and Sanofi.

Author statement

The corresponding author C.C.L. takes full responsibility for the data, the analyses and interpretation, and the conduct of the research; the principal author had full access to all of the data; and has the right to publish any and all data separate and apart from any sponsor.

Supplementary material

Supplementary material is available online.

Funding

This biomarker research study was supported by funds to the Alberta Multiple Sclerosis Collaboration from the Ministry of Economic Development, Trade and Tourism; and the Ministry of Health, of the Government of Alberta. The Cumming Medical Research Fund also supported the biomarker analyses. The clinical trial of HCQ in PPMS (clinicaltrials.gov identifier NCT02913157) was supported through a grant from the MS Translational Clinical Trials Program of the Hotchkiss Brain Institute at the University of Calgary.

Author Contribution

C.C.L. contributed to the conception and design of this study. C.S., N.B., and C.C.L. contributed to the acquisition, storing, and processing of serum samples. Y.Z. contributed to the acquisition of MRI data and statistical analyses in the domperidone study (Clinicaltrials.gov identifier NCT02493049) and M.K. provided samples from the hydroxychloroquine trial, while also editing and revising the manuscript. H.Y. and C.C.L. contributed to the statistical analysis of the study, and drafting of all text, figures, and tables.

Acknowledgement

We would like to thank staff at the University of Calgary Multiple Sclerosis research team for their assistance and support. Biorender® was used to create Figure 7.

Availability of data and materials

We did not have the participants’ consent and research ethics committee approval to share data at the participant level from the initial trial.

Lublin FD, Reingold SC, Cohen JA, Cutter GR, Sorensen PS, Thompson AJ, et al. Defining the clinical course of multiple sclerosis: the 2013 revisions. Neurology. 2014;83(3):278–86.
Miller DH, Leary SM. Primary-progressive multiple sclerosis. Lancet Neurol. 2007;6(10):903–12.
Hawker K, O'Connor P, Freedman MS, Calabresi PA, Antel J, Simon J, et al. Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial. Ann Neurol. 2009;66(4):460–71.
Montalban X, Hauser SL, Kappos L, Arnold DL, Bar-Or A, Comi G, et al. Ocrelizumab versus Placebo in Primary Progressive Multiple Sclerosis. N Engl J Med. 2017;376(3):209–20.
Yong HYF, Yong VW. Mechanism-based criteria to improve therapeutic outcomes in progressive multiple sclerosis. Nat Rev Neurol. 2022;18(1):40–55.
Trapp BD, Stys PK. Virtual hypoxia and chronic necrosis of demyelinated axons in multiple sclerosis. Lancet Neurol. 2009;8(3):280–91.
Lassmann H. The contribution of neuropathology to multiple sclerosis research. Eur J Neurol. 2022;29(9):2869–77.
Gaire BP. Microglia as the Critical Regulators of Neuroprotection and Functional Recovery in Cerebral Ischemia. Cell Mol Neurobiol. 2021.
Girolamo F, Coppola C, Ribatti D, Trojano M. Angiogenesis in multiple sclerosis and experimental autoimmune encephalomyelitis. Acta Neuropathol Commun. 2014;2:84.
Hamid KM, Mirshafiey A. Role of Proangiogenic Factors in Immunopathogenesis of Multiple Sclerosis. Iran J Allergy Asthma Immunol. 2016;15(1):1–12.
Huang J, Khademi M, Fugger L, Lindhe O, Novakova L, Axelsson M, et al. Inflammation-related plasma and CSF biomarkers for multiple sclerosis. Proc Natl Acad Sci U S A. 2020;117(23):12952–60.
D'Haeseleer M, Beelen R, Fierens Y, Cambron M, Vanbinst AM, Verborgh C, et al. Cerebral hypoperfusion in multiple sclerosis is reversible and mediated by endothelin-1. Proc Natl Acad Sci U S A. 2013;110(14):5654–8.
Tottenham I, Koch M, Camara-Lemarroy C. Serum HGF and APN2 are associated with disability worsening in SPMS. J Neuroimmunol. 2022;364:577803.
Kunze R, Marti HH. Angioneurins - Key regulators of blood-brain barrier integrity during hypoxic and ischemic brain injury. Prog Neurobiol. 2019;178:101611.
Kundi S, Bicknell R, Ahmed Z. The role of angiogenic and wound-healing factors after spinal cord injury in mammals. Neurosci Res. 2013;76(1–2):1–9.
Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983;33(11):1444–52.
Koch MW, Sage K, Kaur S, Kim J, Cerchiaro G, Yong VW, et al. Repurposing Domperidone in Secondary Progressive Multiple Sclerosis: A Simon 2-Stage Phase 2 Futility Trial. Neurology. 2021;96(18):e2313–22.
Koch MW, Kaur S, Sage K, Kim J, Levesque-Roy M, Cerchiaro G, et al. Hydroxychloroquine for Primary Progressive Multiple Sclerosis. Ann Neurol. 2021;90(6):940–8.
Motl RW, Cohen JA, Benedict R, Phillips G, LaRocca N, Hudson LD, et al. Validity of the timed 25-foot walk as an ambulatory performance outcome measure for multiple sclerosis. Mult Scler. 2017;23(5):704–10.
Herbers J, Miller R, Walther A, Schindler L, Schmidt K, Gao W, et al. How to deal with non-detectable and outlying values in biomarker research: Best practices and recommendations for univariate imputation approaches. Compr Psychoneuroendocrinol. 2021;7:100052.
Levy YA, Fainberg KM, Amidror T, Regev K, Auriel E, Karni A. High and dysregulated secretion of epidermal growth factor from immune cells of patients with relapsing-remitting multiple sclerosis. J Neuroimmunol. 2013;257(1–2):82–9.
Tejera-Alhambra M, Casrouge A, de Andres C, Seyfferth A, Ramos-Medina R, Alonso B, et al. Plasma biomarkers discriminate clinical forms of multiple sclerosis. PLoS ONE. 2015;10(6):e0128952.
Scalabrino G. New Epidermal-Growth-Factor-Related Insights Into the Pathogenesis of Multiple Sclerosis: Is It Also Epistemology? Front Neurol. 2021;12:754270.
Holley JE, Newcombe J, Whatmore JL, Gutowski NJ. Increased blood vessel density and endothelial cell proliferation in multiple sclerosis cerebral white matter. Neurosci Lett. 2010;470(1):65–70.
Karampoor S, Zahednasab H, Ramagopalan S, Mehrpour M, Keyvani H. Angiogenic factors are associated with multiple sclerosis. J Neuroimmunol. 2016;301:88–93.
Thone J, Kleiter I, Stahl A, Ellrichmann G, Gold R, Hellwig K. Relevance of endoglin, IL-1alpha, IL-1beta and anti-ovarian antibodies in females with multiple sclerosis. J Neurol Sci. 2016;362:240–3.
Park ES, Kim S, Yao DC, Savarraj JPJ, Choi HA, Chen PR et al. Soluble Endoglin Stimulates Inflammatory and Angiogenic Responses in Microglia That Are Associated with Endothelial Dysfunction. Int J Mol Sci. 2022;23(3).
Shen F, Degos V, Chu PL, Han Z, Westbroek EM, Choi EJ, et al. Endoglin deficiency impairs stroke recovery. Stroke. 2014;45(7):2101–6.
Zhu W, Ma L, Zhang R, Su H. The roles of endoglin gene in cerebrovascular diseases. Neuroimmunol Neuroinflamm. 2017;4:199–210.
Urshansky N, Mausner-Fainberg K, Auriel E, Regev K, Karni A. Low and dysregulated production of follistatin in immune cells of relapsing-remitting multiple sclerosis patients. J Neuroimmunol. 2011;238(1–2):96–103.
Morianos I, Trochoutsou AI, Papadopoulou G, Semitekolou M, Banos A, Konstantopoulos D, et al. Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-alpha-dependent pathways. Proc Natl Acad Sci U S A. 2020;117(22):12269–80.
Dillenburg A, Ireland G, Holloway RK, Davies CL, Evans FL, Swire M, et al. Activin receptors regulate the oligodendrocyte lineage in health and disease. Acta Neuropathol. 2018;135(6):887–906.
Holloway RK, Ireland G, Sullivan G, Becher JC, Smith C, Boardman JP, et al. Microglial inflammasome activation drives developmental white matter injury. Glia. 2021;69(5):1268–80.
Kitamura K, Iwanami A, Nakamura M, Yamane J, Watanabe K, Suzuki Y, et al. Hepatocyte growth factor promotes endogenous repair and functional recovery after spinal cord injury. J Neurosci Res. 2007;85(11):2332–42.
Bai L, Lennon DP, Caplan AI, DeChant A, Hecker J, Kranso J, et al. Hepatocyte growth factor mediates mesenchymal stem cell-induced recovery in multiple sclerosis models. Nat Neurosci. 2012;15(6):862–70.
Schenk GJ, Dijkstra S, van het Hof AJ, van der Pol SM, Drexhage JA, van der Valk P, et al. Roles for HB-EGF and CD9 in multiple sclerosis. Glia. 2013;61(11):1890–905.
Ashok A, Rai NK, Raza W, Pandey R, Bandyopadhyay S. Chronic cerebral hypoperfusion-induced impairment of Abeta clearance requires HB-EGF-dependent sequential activation of HIF1alpha and MMP9. Neurobiol Dis. 2016;95:179–93.
Marrodan M, Farez MF, Balbuena Aguirre ME, Correale J. Obesity and the risk of Multiple Sclerosis. The role of Leptin. Ann Clin Transl Neurol. 2021;8(2):406–24.
Loonstra FC, Falize KF, de Ruiter LRJ, Schoonheim MM, Strijbis EMM, Killestein J, et al. Adipokines in multiple sclerosis patients are related to clinical and radiological measures. J Neurol. 2023;270(4):2018–30.
Xie XF, Huang XH, Shen AZ, Li J, Sun YH. Association between circulating leptin levels and multiple sclerosis: a systematic review and meta-analysis. Postgrad Med J. 2018;94(1111):278–83.
Biernacki T, Kokas Z, Sandi D, Fuvesi J, Fricska-Nagy Z, Farago P et al. Emerging Biomarkers of Multiple Sclerosis in the Blood and the CSF: A Focus on Neurofilaments and Therapeutic Considerations. Int J Mol Sci. 2022;23(6).
Batocchi AP, Rotondi M, Caggiula M, Frisullo G, Odoardi F, Nociti V, et al. Leptin as a marker of multiple sclerosis activity in patients treated with interferon-beta. J Neuroimmunol. 2003;139(1–2):150–4.
Bistrom M, Hultdin J, Andersen O, Alonso-Magdalena L, Jons D, Gunnarsson M, et al. Leptin levels are associated with multiple sclerosis risk. Mult Scler. 2021;27(1):19–27.
Kirk SL, Karlik SJ. VEGF and vascular changes in chronic neuroinflammation. J Autoimmun. 2003;21(4):353–63.
Claudio L, Raine CS, Brosnan CF. Evidence of persistent blood-brain barrier abnormalities in chronic-progressive multiple sclerosis. Acta Neuropathol. 1995;90(3):228–38.
Proescholdt MA, Jacobson S, Tresser N, Oldfield EH, Merrill MJ. Vascular endothelial growth factor is expressed in multiple sclerosis plaques and can induce inflammatory lesions in experimental allergic encephalomyelitis rats. J Neuropathol Exp Neurol. 2002;61(10):914–25.
Graumann U, Reynolds R, Steck AJ, Schaeren-Wiemers N. Molecular changes in normal appearing white matter in multiple sclerosis are characteristic of neuroprotective mechanisms against hypoxic insult. Brain Pathol. 2003;13(4):554–73.
Kirk J, Plumb J, Mirakhur M, McQuaid S. Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood-brain barrier leakage and active demyelination. J Pathol. 2003;201(2):319–27.
Masuda H, Mori M, Uzawa A, Uchida T, Muto M, Ohtani R, et al. Elevated serum levels of bone morphogenetic protein-9 are associated with better outcome in AQP4-IgG seropositive NMOSD. Sci Rep. 2023;13(1):3538.
Graves JS, Krysko KM, Hua LH, Absinta M, Franklin RJM, Segal BM. Ageing and multiple sclerosis. Lancet Neurol. 2023;22(1):66–77.
Ungvari Z, Tarantini S, Kiss T, Wren JD, Giles CB, Griffin CT, et al. Endothelial dysfunction and angiogenesis impairment in the ageing vasculature. Nat Rev Cardiol. 2018;15(9):555–65.
Kirk S, Frank JA, Karlik S. Angiogenesis in multiple sclerosis: is it good, bad or an epiphenomenon? J Neurol Sci. 2004;217(2):125–30.
Lange C, Storkebaum E, de Almodovar CR, Dewerchin M, Carmeliet P. Vascular endothelial growth factor: a neurovascular target in neurological diseases. Nat Rev Neurol. 2016;12(8):439–54.
Hiratsuka D, Kurganov E, Furube E, Morita M, Miyata S. VEGF- and PDGF-dependent proliferation of oligodendrocyte progenitor cells in the medulla oblongata after LPC-induced focal demyelination. J Neuroimmunol. 2019;332:176–86.
Macmillan CJ, Starkey RJ, Easton AS. Angiogenesis is regulated by angiopoietins during experimental autoimmune encephalomyelitis and is indirectly related to vascular permeability. J Neuropathol Exp Neurol. 2011;70(12):1107–23.
Li Z, Korhonen EA, Merlini A, Strauss J, Wihuri E, Nurmi H, et al. Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS. J Clin Invest. 2020;130(4):1977–90.
Liu XS, Chopp M, Zhang RL, Hozeska-Solgot A, Gregg SC, Buller B, et al. Angiopoietin 2 mediates the differentiation and migration of neural progenitor cells in the subventricular zone after stroke. J Biol Chem. 2009;284(34):22680–9.
Aguirre A, Dupree JL, Mangin JM, Gallo V. A functional role for EGFR signaling in myelination and remyelination. Nat Neurosci. 2007;10(8):990–1002.
Desole C, Gallo S, Vitacolonna A, Montarolo F, Bertolotto A, Vivien D, et al. HGF and MET: From Brain Development to Neurological Disorders. Front Cell Dev Biol. 2021;9:683609.
Schnitzler AC, Mellott TJ, Lopez-Coviella I, Tallini YN, Kotlikoff MI, Follettie MT, et al. BMP9 (bone morphogenetic protein 9) induces NGF as an autocrine/paracrine cholinergic trophic factor in developing basal forebrain neurons. J Neurosci. 2010;30(24):8221–8.
Fujita Y, Yamashita T. The Effects of Leptin on Glial Cells in Neurological Diseases. Front Neurosci. 2019;13:828.
Rezabakhsh A, Montazersaheb S, Nabat E, Hassanpour M, Montaseri A, Malekinejad H, et al. Effect of hydroxychloroquine on oxidative/nitrosative status and angiogenesis in endothelial cells under high glucose condition. Bioimpacts. 2017;7(4):219–26.
Rahman R, Murthi P, Singh H, Gurusinghe S, Mockler JC, Lim R, et al. The effects of hydroxychloroquine on endothelial dysfunction. Pregnancy Hypertens. 2016;6(4):259–62.
Dong Y, Lu Y, Xia Y, Wang X. Effect of hydroxychloroquine on antiphospholipid antibodies-inhibited endometrial angiogenesis. J Matern Fetal Neonatal Med. 2021:1–9.
Schreiber K, Breen K, Parmar K, Rand JH, Wu XX, Hunt BJ. The effect of hydroxychloroquine on haemostasis, complement, inflammation and angiogenesis in patients with antiphospholipid antibodies. Rheumatology (Oxford). 2018;57(1):120–4.
Toledo FGS, Miller RG, Helbling NL, Zhang Y, DeLany JP. The effects of hydroxychloroquine on insulin sensitivity, insulin clearance and inflammation in insulin-resistant adults: A randomized trial. Diabetes Obes Metab. 2021;23(6):1252–61.
Koch MW, Zabad R, Giuliani F, Hader W Jr., Lewkonia R, Metz L, et al. Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis. J Neurol Sci. 2015;358(1–2):131–7.
Ma Y, Yang S, He Q, Zhang D, Chang J. The Role of Immune Cells in Post-Stroke Angiogenesis and Neuronal Remodeling: The Known and the Unknown. Front Immunol. 2021;12:784098.

No competing interests reported.

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Angiogenesis biomarkers discriminate multiple sclerosis phenotypes

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Abstract

Figures

Introduction

Materials and methods

Study design and participants

Angiogenesis markers

Outcomes

Analysis and descriptive statistics

Ethics

Results

Baseline characteristics and circulating angiogenesis markers

Circulating angiogenesis markers are dysregulated across MS phenotypes and correlate with clinical disability

Circulating angiogenesis markers correlate with inflammatory activity

Transcriptomic changes are consistent with angiogenesis dysregulation in the MS CNS

Angiogenic differences in MS demyelinating lesions compared to MS normal appearing brain or controls

Angiogenic differences across MS phenotypes and brain regions

Angioneurins are associated with remyelination and disability outcomes in clinical trial cohorts

Discussion

Angiogenesis markers are dysregulated in the serum/CNS in MS, and can discriminate phenotypes

Epidermal growth factor (EGF)

Endoglin

Follistatin

Hepatocyte growth factor (HGF)

Heparin binding-epidermal growth factor (HB-EGF)

Leptin

Angiogenesis markers have relevance for resilience against injury and the effect of age

Angiogenesis markers play complex roles in CNS injury

Hydroxychloroquine (HCQ) may modify angiogenic factors in PPMS

Limitations

Conclusion

Declarations

Ethics approval and consent to participate

Competing interests

Author statement

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Supplementary Files

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