Hydroxychloroquine modifies angiogenesis factors in primary progressive multiple sclerosis

doi:10.21203/rs.3.rs-2129915/v1

Download PDF

Research Article

Hydroxychloroquine modifies angiogenesis factors in primary progressive multiple sclerosis

https://doi.org/10.21203/rs.3.rs-2129915/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

In this study we investigated serum angiogenic markers of hydroxychloroquine (HCQ)-treated patients with primary-progressive multiple sclerosis (PPMS) from a phase II clinical trial. PPMS patients had higher levels of endothelin-1 (ETN-1), fibroblast growth factor-2 (FGF-2), granulocyte-colony stimulating factor (G-CSF), heparin binding epidermal-growth factor, and interleukin-8 compared to healthy controls. At 6 months, the angiogenic markers ETN-1, FGF-2, G-CSF, and vascular endothelial growth factor-A were significantly reduced; conversely, angiopoietin-2, endoglin, and leptin were increased. Angiogenic changes did not correlate with disability worsening, however changes were more profound in stable patients suggesting a possible treatment effect with HCQ.

Multiple Sclerosis

PPMS

progressive multiple sclerosis

angiogenesis

hydroxychloroquine

Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the central nervous system (CNS). Primary progressive MS (PPMS), the least common form of MS, presents as steadily increasing unremitting disability from disease onset (Miller and Leary 2007). Immune-modulating drugs used commonly in the relapsing form(s) of MS have demonstrated only modest effects on PPMS disability accumulation. When analyzing PPMS cohorts and the effects of immune-modulating therapy, the greatest benefit is observed in those with some form of inflammatory activity; this includes enhancing lesions on magnetic resonance imaging (MRI) or with recent relapses (Hawker, O'Connor et al. 2009, Montalban, Hauser et al. 2017). Progression in the absence of inflammatory disease activity is far more difficult to treat, perhaps due to different pathophysiology on a molecular and cellular level (Yong and Yong 2022).

One of several pathophysiological mechanisms suggested to have a prominent role in progressive MS is microglial/macrophage over-representation and activation correlating to ongoing demyelination and neuronal/axonal loss (Yong and Yong 2022). In the context of cerebral ischemia, microglia/macrophage secrete reactive oxygen/nitrogen species contributing to oxidative stress and leading to a state of virtual hypoxia (Trapp and Stys 2009, Yong and Yong 2022). Thus, microglia/macrophage may regulate proangiogenic factors, where angiogenesis is described as the formation of new blood vessels from pre-existing ones (Gaire 2021). An altered balance in angiogenic molecules has been shown to relate both to chronic inflammation and to neurodegeneration (Girolamo, Coppola et al. 2014, Hamid and Mirshafiey 2016). Angiogenic molecules, notably endothelin-1 and vascular endothelial growth factor, are increased in people with MS compared to controls (D'Haeseleer, Beelen et al. 2013, Girolamo, Coppola et al. 2014, Hamid and Mirshafiey 2016, Huang, Khademi et al. 2020), and are increased in patients with secondary progressive MS who show disability worsening (Tottenham, Koch et al. 2022). Although angiogenesis is only one part of the complex pathogenesis of MS, it has been postulated as a promising target for therapeutics (Fig. 1) (Girolamo, Coppola et al. 2014).

Hydroxychloroquine (HCQ) is a repurposed antimalarial drug commonly used for the treatment of rheumatoid arthritis and systemic lupus erythematous. It crosses the blood-brain barrier, has been shown to reduce microglial activation in vitro and in an animal model of MS (Koch, Zabad et al. 2015), inhibits oxidative stress and neuronal killing (Yong and Yong 2022), and may contribute to angiogenesis regulation (Fig. 1). In vitro HCQ reduces angiogenesis in cultured endothelial cells (Rahman, Murthi et al. 2016, Rezabakhsh, Montazersaheb et al. 2017, Dong, Lu et al. 2021) and mitigates vascular dysfunction (Rahman, Murthi et al. 2020). In a phase II clinical trial of inactive PPMS (clinicaltrials.gov identifier NCT02913157), treatment with HCQ reduced the expected rate of disability worsening at 18 months (Koch, Kaur et al. 2021).

We hypothesized that markers of angiogenesis would be altered in the serum of PPMS, and that levels would decrease over time with HCQ treatment. We tested this hypothesis by comparing the longitudinal trajectories of angiogenesis molecules in HCQ-treated patients with PPMS from the original Koch et al. trial (Koch, Kaur et al. 2021). We then investigated the association of angiogenesis markers and clinical variables.

2.1 Study Design and Participants

Serum samples were obtained from all 39 participants in the single-arm Phase II futility trial of 200mg oral HCQ twice daily for 18 months (clinicaltrials.gov identifier NCT02913157) (Koch, Kaur et al. 2021). 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. The primary outcome of the futility trial was ≥ 20% worsening on the timed 25-foot walk (T25FW) (Motl, Cohen et al. 2017) between 6 and 18 months of follow-up. Based on original trial data, 40% of the cohort were expected to worsen; however, only 22.9% (9 out of 39) did so, and HCQ was deemed to be non-futile. Full inclusion and exclusion criteria can be found in the original publication (Koch, Kaur et al. 2021). Serum samples were also collected from 10 healthy controls with no history of inflammatory, neurodegenerative, gastrointestinal disease, or treatment with immune-modulating therapy. Controls were appropriately matched to the PPMS cohort by age and sex. Serum samples from both cohorts were drawn at baseline (month 0) and month 6 to allow for HCQ to become effective and show a potential treatment effect.

2.2 Study Ethics

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

2.3 Angiogenesis Markers

Blood samples were drawn from participants using a standard peripheral venipuncture by an experienced phlebotomist at month 0 and 6. Serum was isolated by centrifugation at 3000×g for 10-min, and samples were aliquoted and stored at − 80°C until analyzed. The Human Angiogenesis Array & Growth Factor Array® multiplex (Eve Technologies, Calgary, Canada) was used to measure angiogenesis factors. Proteins tested in this array included angiopoietin-2, bone morphogenetic protein-9, epidermal growth factor, endoglin, endothelin-, fibroblast growth factor-1 and 2, follistatin, granulocyte-colony stimulating factor, heparin binding-epidermal growth factor, hepatocyte growth factor, interleukin-8, leptin, placental growth factor, vascular endothelial cell growth factor-A, C, and D. Details of the multiplex method and limits of detection of this assay are available online at https://www.evetechnologies.com/product/human-angiogenesis-array-growth-factor-17-plex-array/.

2.4 Outcomes

Primary outcomes included comparing angiogenic markers between PPMS patients and healthy controls at baseline; assessing changes in angiogenic markers at 6 months while on HCQ; and whether any angiogenesis changes correlated with reduction in disability progression as measured using retinal nerve fiber layer (RNFL) thickness (see original trial for methods of obtaining RNFL measurements) (Koch, Kaur et al. 2021), T25FW(Motl, Cohen et al. 2017), and the expanded disability status scale (EDSS).

2.5 Analysis and Descriptive Statistics

Baseline characteristics were summarized using frequency (percent) for categorical variables and mean ± standard deviation (SD) 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 divided according to progression based on the T25FW test (worsening ≥ 20%) at 12-months in the original trial (Koch, Kaur et al. 2021). Groups were compared using the Student’s t-test, Mann–Whitney U test, one-way ANOVA, Kruskal-Wallis test, or Chi square test using the Statistical Package for Social Sciences (SPSS 22.0, IL, USA) and GraphPad PRISM (PRISM 9.0, GraphPad Software, USA). To investigate the prognostic potential of the biomarkers on outcome (T25FW worsening from month 6 to month 18), baseline age, sex, EDSS, T25Wt, RNFL thickness and baseline neurofilament and glial fibrillary acidic protein were explored using the univariate Mann-Whitney test. Significant variables were then included in a multivariate binary logistic regression model with outcome as the dependent variable. The underlying assumptions of the binary logistic regression models were acceptable in the obtained plots.

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 50% of values outside of range were excluded from analysis. 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. A Bonferroni correction was used to adjust the multiplex analyses for multiple comparisons. Multivariate logistic regression analysis with worsening in T25FW as the dichotomous target variable was carried out including significantly different variables along with age and baseline T25FW score.

3.1 Patient Characteristics

The initial phase II trial was conducted from Nov 17 2016 to June 4 2021 at the Calgary MS Clinic in Calgary, Alberta, Canada (Koch, Kaur et al. 2021). Thirty-nine patients were included in the primary outcome analysis and at 18 months. Nine (23%) of the 39 trial participants met primary endpoint of disability worsening (Koch, Kaur et al. 2021). All 39 trial participants were included in this study and age/gender matched to 10 controls. Their clinical and demographic characteristics are outlined in Table 1.

Table 1

**Baseline characteristics of the PPMS patients treated with hydroxychloroquine (n = 39) compared to controls (n = 10).** The baseline characteristics of the PPMS cohort are separated based on whether primary outcome in the phase II trial (Koch, Kaur et al. 2021) was achieved at month 18. EDSS = Expanded Disability Status Scale, HCQ = hydroxychloroquine, MS = Multiple Sclerosis, N = number, SDMT = Symbol Digit Modalities Test, RNFL = Retinal Nerve Fibre Layer, T25FW = Timed 25-Foot Walk Test.
	Controls (n = 10)	PPMS patients treated with HCQ (n = 39)	PPMS patients treated with HCQ who were stable at 18 months (n = 30)	PPMS patients treated with HCQ who met primary outcome for disability worsening at 18 months (n = 9)
Age, years	55.9 ± 6	55.6 ± 6.1	55.46 ± 5.8	56 ± 7.3
n (%) female	6 (60%)	15 (38.4)	12 (40)	3 (33.3)
EDSS (range)	NA	6 (4-6.5)	6 (4-6.5)	6.5 (4-6.5)
T25FW, seconds (range)	NA	8 (5.3–39.7)	7.4 (5.3–39.7)	12.6 (5.5–24)**
SDMT	NA	52.1 ± 10.5	52.2 ± 10.7	51.7 ± 10
RNFL thickness, µm Missing values: 1	NA	84.25 ± 9.88	86.2 ± 9.7	77.8 ± 7.7*
Disease duration, years (range)	NA	7.5 (2-25.2)	8.2 (2-25.2)	7.1 (2.8–17.9)

*=p < 0.05 vs. stable

**=p < 0.01 vs. stable

3.2 Angiogenesis Markers at Baseline

The angiogenesis markers endothelin-1 (p = 0.0015), fibroblast growth factor-2 (p = 0.0015), granulocyte-colony stimulating factor (p = 0.0015), heparin binding-epidermal growth factor (p = 0.0075) and interleukin-8 (p = 0.0015) were significantly higher in people living with PPMS compared to controls (Fig. 2). The markers fibroblast growth factor-1 and placental growth factor were excluded from the analysis as levels were out-of-range (below the detectable limit) at baseline. The rest of the biomarkers were comparable at baseline between people with PPMS and healthy controls (Fig. 2). Concentrations of leptin were significantly higher in females compared to males (65,404 ± 64,795 vs 17,538 ± 15,732, p = 0.015); other molecules did not separate based on gender.

In analyzing markers of angiogenesis at baseline with clinical phenotype, none of them correlated with baseline age, EDSS, disease duration, SDMT scores, RNFL, or T25FW. The biomarkers at baseline were also not associated with disability progression at end-of-study (not shown); thus, logistic regression models were not performed.

3.3 The Effect of Hydroxychloroquine on Angiogenesis Markers Based on Longitudinal Dynamics

The angiogenic molecules endothelin-1 (-25pg/mL [-22.5%, p = 0.0015]), fibroblast growth factor-2 (− 24 pg/mL [-13%, p = 0.0015]), granulocyte-colony stimulating factor (-47 pg/mL [-30.7%, p = 0.0015]), and vascular endothelial growth factor-A (-120 pg/mL [-22.1%, p = 0.045]) were significantly reduced at 6 months in patients with PPMS treated with HCQ compared to baseline (Fig. 3). The angiogenic molecules angiopoietin-2 (+ 967 pg/mL [+ 17%, p = 0.045]), endoglin (+ 381 pg/mL [17.9%, p = 0.0015]) and leptin (+ 17,447 pg/mL [+ 48.5%, p = 0.015]) were significantly increased (Fig. 4). The rest of the biomarkers were similar between groups.

3.4 Angiogenesis Regulation by Hydroxychloroquine in PPMS appears to Correlate with Disability Progression

We compared the longitudinal dynamics of angiogenesis markers at month 6 between PPMS participants treated with HCQ who met the primary outcome (disability progression, n = 9) and those who remained stable (n = 30). Although angiopoietin-2, endoglin, endothelin-1, fibroblast growth factor-2, granulocyte-colony stimulating factor, leptin, and vascular endothelial growth factor-A displayed significant changes from baseline to month 6 (Fig. 3, 4), this change did not correlate with clinical disability worsening. Interestingly, in stable patients on HCQ, and not so in the non-responders, there was reduction in endothelin-1 (p = 0.0015), fibroblast growth factor-2 (p = 0.0015), and granulocyte-colony stimulating factor (p = 0.06) over 6 months, while endoglin was increased (p = 0.021) (Fig. 5).

4.1 Angiogenesis may play dichotomous roles in multiple sclerosis and other inflammatory disorders

Angiogenesis, the formation of new blood vessels, is generally quiescent in healthy adults with the exception of strictly controlled physiologic situations such as female reproduction, and tissue repair (Kirk, Frank et al. 2004). Angiogenesis allows access to increased oxygen and nutrients in areas of increased cellular needs; however, this advantageous process can be disrupted in diseases where chronic inflammation and a hypoxic environment are present leading to over-activation of angiogenic factors (Kirk, Frank et al. 2004). MS is one such disease fueled by chronic inflammation, neurodegeneration, and the creation of a virtually hypoxic CNS environment (Yong and Yong 2022). Uncontrolled angiogenesis serves to increase vascular permeability and compromise the blood-brain barrier, and transport inflammatory cells, nutrients, and oxygen to the site of inflammation – together this perpetuates disease activity (Fig. 1) (Claudio, Raine et al. 1995, Kirk and Karlik 2003, Girolamo, Coppola et al. 2014). Dysregulation of angiogenic factors is present in MS as demonstrated by the increased number of angiogenic factors present in MS lesions when compared to controls; and by the amelioration of disease activity in animal models of MS using anti-angiogenesis agents (Claudio, Raine et al. 1995, Proescholdt, Jacobson et al. 2002, Graumann, Reynolds et al. 2003, Kirk, Plumb et al. 2003, Girolamo, Coppola et al. 2014).

Several specific angiogenesis factors are increased in MS pathophysiology. Vascular endothelial growth factor-A is expressed in multiple sclerosis plaques and can induce inflammation in experimental models of MS (Proescholdt, Heiss et al. 1999, Proescholdt, Jacobson et al. 2002, Girolamo, Coppola et al. 2014). Fibroblast growth factor-2 is dramatically increased in progressive demyelination models with strongest expression on macrophages/microglia (Sarchielli, Di Filippo et al. 2008). Endothelin-1 is a potent vasoconstrictor that is thought to aggravate cerebral hypoperfusion in MS leading to a state of virtual hypoxia and neuron degeneration (Speciale, Sarasella et al. 2000, Haufschild, Shaw et al. 2001, Pache, Kaiser et al. 2003, Girolamo, Coppola et al. 2014); and endothelin-1 antagonism ameliorates disease in animal models of MS (Shin, Kang et al. 2001). Blood levels of endothelin-1 are higher in people living with MS (D'Haeseleer, Beelen et al. 2013, Monti, Morbidelli et al. 2017, Castellazzi, Lamberti et al. 2019, Rocha, Colpo et al. 2019), with the highest concentrations found in progressive forms of the disease (Monti, Morbidelli et al. 2017). Heparin binding-epidermal growth factor appears to hinder cellular migration and adhesion, and is thought to play a process in lesion formation demonstrated by higher concentrations in activated astrocytes in MS lesions (Schenk, Dijkstra et al. 2013). Similarly, granulocyte-colony stimulating factor has been implicated for its pro-inflammatory effects on macrophage and microglia (Openshaw, Stuve et al. 2000, Snir, Lavie et al. 2006, Ifergan, Davidson et al. 2017). Interleukin-8 has been associated with an inflammatory phenotype in MS (Donninelli, Studer et al. 2021), acts indirectly to upregulate matrix metalloproteinases to alter the extracellular matrix (Hamid and Mirshafiey 2016), and there is evidence of intrathecal production of interluekin-8 in PPMS (Ukkonen, Wu et al. 2007). In patients with more severe disability, the production of cytokines such as granulocyte-colony stimulating factor and interleukin-8 appears to be less coordinated due to advanced neurodegenerative disease (Donninelli, Studer et al. 2021). In our study, the molecules endothelin-1, heparin binding-epidermal growth factor, granulocyte-colony stimulating factor, and interleukin-8 were significantly elevated in people living with PPMS as compared to controls at baseline; this is consistent with the literature.

It is important to note that although angiogenesis appears to have a detrimental effect in chronic inflammatory diseases, this is complicated by the dual roles that angiogenic factors may play in neurodegeneration and neuroinflammation. For example, fibroblast growth factor-2 has also been implicated in directly stimulating the localized proliferation of oligodendrocytes within MS lesions suggesting a remyelinating component (Sarchielli, Di Filippo et al. 2008). Similarly, vascular endothelial growth factor has been shown to have remyelinating and neuroprotective properties (Lange, Storkebaum et al. 2016, Hiratsuka, Kurganov et al. 2019), and may act as a neuroprotective agent in the late phases of MS (Girolamo, Coppola et al. 2014). Further, people with progressive MS have lower levels of vascular endothelial growth factor-A mRNA transcripts than healthy controls (Rasol, Helmy et al. 2016). Angiopoietin-2 plays a role in acute inflammation in animal models of MS, but increases in concentration in late stages and improves repair after CNS injury (Liu, Chopp et al. 2009, Macmillan, Starkey et al. 2011, Li, Korhonen et al. 2020).

The dichotomous role of angiogenic molecules in PPMS highlights the complex 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 is likely not to elucidate precise mechanisms and their effect in PPMS pathophysiology. Instead, several molecules should be explored simultaneously, as well as their complex interaction with their cellular sources (glia, monocytes, endothelial cells).

4.2 The effect of hydroxychloroquine on angiogenic factors in multiple sclerosis and possible mechanism of action

In our study we found that HCQ leads to significant reduction in certain angiogenic molecules at 6 months, specifically endothelin-1, fibroblast growth factor-2, G-CSF, and vascular endothelial growth factor-A. Serum of HCQ-treated individuals show significant increases in angiopoietin-2, endoglin and leptin. There is literature suggesting that HCQ may modulate angiogenesis. In vitro, HCQ is able to reduce angiogenesis in cultured endothelial cells (Rezabakhsh, Montazersaheb et al. 2017) and it inhibits the production of endothelin-1 in endothelial cells exposed to eclamptic sera (Rahman, Murthi et al. 2016). T-cells treated with HCQ exhibit reductions in granulocyte macrophage-colony stimulating factor expression following inflammatory cytokine stimulation (Kim, Martin et al. 2018). HCQ’s effect on vascular endothelial growth factor-A remains uncertain; in one study HCQ reduced its expression in endothelial cells in vitro (Dong, Lu et al. 2021), however, another small study did not show any changes in patients with antiphospholipid syndrome (Schreiber, Breen et al. 2018). HCQ improves insulin sensitivity and glucose disposition in skeletal muscle (Toledo, Miller et al. 2021), which may account for its increase of the insulin-specific angiogenic marker leptin in our study. Finally, HCQ has been shown to improve vascular dysfunction in pre-eclampsia by mitigating the production of soluble placental-derived anti-angiogenic factors including endoglin (Rahman, Murthi et al. 2020).

Interestingly, when comparing the significant changes in angiogenic markers by month 6 on HCQ, only those patients who were stable had significant angiogenic changes. This suggests that changes in angiogenic molecules may be reflective of HCQ´s biological/therapeutic effect although the clinical significance of such changes remains to be elucidated.

4.3 Possible mechanisms for which hydroxychloroquine affects angiogenic factors

The mechanism by which HCQ regulates angiogenesis is still unknown. It could have direct effects on angiogenesis by directly regulating angiogenic factors as described above. It could also have indirect effects on angiogenesis by decreasing the activation of immune cells such as microglia (Koch, Zabad et al. 2015, Gaire 2021, Ma, Yang et al. 2021), or other CNS cells. Microglia are a predominant cell type in the pathophysiology of progressive MS, and contribute to a hypoxic environment through the production of reactive oxide/nitric species (Yong and Yong 2022). By regulating microglial activation, HCQ could indirectly modify angiogenesis by decreasing chronic inflammation and ameliorating the hypoxic environment seen in progressive MS. We have only limited evidence to speculate that HCQ may be important in preventing disability progression; however it does fulfill many criteria necessary for treatment in PPMS including the ability to penetrate the blood-brain barrier, inhibit microglial and B-cell activity, and interfere with neuronal-killing (Koch, Zabad et al. 2015, Yong and Yong 2022). The exact mechanisms by which HCQ affects angiogenesis and MS remains to be elucidated, and whether angiogenesis markers could be used as indirect markers of HCQ´s effects on microglia, or on any therapeutic effect, warrants further study.

4.4 Limitations and Conclusions

Limitations of this study include the absence of a control/placebo group for the HCQ treatment trial. Although serum samples were matched by age/gender to healthy controls, this does not allow for unequivocal evidence of a treatment effect or for the determination of the biomarker´s natural trajectory in the population studied. Another limitation of our study is the relatively small sample size (n = 9 compared to n = 30 in the original trial) and short-term follow-up (6 months compared to 18 months in the original try) limited secondary to serum storage and funding. The trajectory of the biomarkers at further follow-up, as well as any correlation with disability progression, remain unknown. Finally, the angiogenesis marker analysis was done in serum, and it is unclear if this is reflective of what may be occurring in the CNS/cerebrospinal fluid in PPMS. More studies are needed to elucidate the roles of angiogenesis in neurodegeneration and neuroinflammation.

In conclusion, angiogenic markers are dysregulated in the progressive stages of MS in this serum-study of PPMS patients treated with HCQ. HCQ appears to ameliorate potent angiogenic factors that can contribute to neuroinflammation and neurodegeneration, potentially through microglial mechanisms. Furthermore, the effect was more pronounced in participants who remained stable over the course of the study. This suggests that changes in angiogenic molecules may be reflective of HCQ´s biological/therapeutic effect. More studies are needed to fully elucidate the significance of these results.

CNS= Central nervous system

HCQ= Hydroxychloroquine

EDSS= Expanded disability status scale

MRI= Magnetic resonance imaging

MS= Multiple sclerosis

PPMS= Primary progressive multiple sclerosis

T25FW= Timed 25-foot walk

RNFL= Retinal nerve fibre layer

GENES

APN2= Angiopoietin-2

BMP9= Bone morphogenetic protein-9

EGF= Epidermal growth factor

ENG= Endoglin

ETN-1= Endothelin-1

FGF-1/2= Fibroblast growth factor

FST= Follistatin

G-CSF= Granulocyte-colony stimulating factor

HB-EGF= Heparin binding-epidermal growth factor

HGF= Hepatocyte growth factor

IL-8= Interleukin (IL)-8,

LEP= Leptin

PLGF= Placental growth factor

VEGF-A/C/D= Vascular endothelial growth factor

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.

Acknowledgements

We would like to thank staff at the University of Calgary Multiple Sclerosis research team for their assistance and support.

Author Contributions

M.W.K., and C. L. contributed to the conception and design of this study. I.T., C.S., and C.L. contributed to the acquisition of serum samples. H.Y. and C.L. contributed to the statistical analysis of the study, and drafting of all text, figures, and tables.

Statements and Declarations.

The corresponding author 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.H.Y., I.T. and C.S. have nothing to disclose. M.W.K received consulting fees and travel support from Biogen, Novartis, Roche, Sanofi Genzyme, and EMD Serono, outside of this work. C.L. received consult fees from EMD Serono, outside of this work.

Data availability

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

Castellazzi M et al (2019) "Increased Levels of Endothelin-1 in Cerebrospinal Fluid Are a Marker of Poor Visual Recovery after Optic Neuritis in Multiple Sclerosis Patients." Dis Markers 2019: 9320791
Claudio L et al (1995) "Evidence of persistent blood-brain barrier abnormalities in chronic-progressive multiple sclerosis". Acta Neuropathol 90(3):228–238
D'Haeseleer M et al (2013) "Cerebral hypoperfusion in multiple sclerosis is reversible and mediated by endothelin-1". Proc Natl Acad Sci U S A 110(14):5654–5658
Dong Y et al (2021) "Effect of hydroxychloroquine on antiphospholipid antibodies-inhibited endometrial angiogenesis."J Matern Fetal Neonatal Med:1–9
Donninelli G et al (2021) "Immune Soluble Factors in the Cerebrospinal Fluid of Progressive Multiple Sclerosis Patients Segregate Into Two Groups. " Front Immunol 12:633167
Gaire BP (2021) "Microglia as the Critical Regulators of Neuroprotection and Functional Recovery in Cerebral Ischemia. " Cell Mol Neurobiol
Girolamo F et al (2014) Angiogenesis in multiple sclerosis and experimental autoimmune encephalomyelitis. Acta Neuropathol Commun 2:84
Graumann U et al (2003) "Molecular changes in normal appearing white matter in multiple sclerosis are characteristic of neuroprotective mechanisms against hypoxic insult". Brain Pathol 13(4):554–573
Hamid KM, Mirshafiey A (2016) "Role of Proangiogenic Factors in Immunopathogenesis of Multiple Sclerosis". Iran J Allergy Asthma Immunol 15(1):1–12
Haufschild T et al (2001) "Increased endothelin-1 plasma levels in patients with multiple sclerosis". J Neuroophthalmol 21(1):37–38
Hawker K et al (2009) "Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial". Ann Neurol 66(4):460–471
Hiratsuka D et al (2019) "VEGF- and PDGF-dependent proliferation of oligodendrocyte progenitor cells in the medulla oblongata after LPC-induced focal demyelination". J Neuroimmunol 332:176–186
Huang J et al (2020) Inflammation-related plasma and CSF biomarkers for multiple sclerosis. Proc Natl Acad Sci U S A 117(23):12952–12960
Ifergan I et al (2017) "Targeting the GM-CSF receptor for the treatment of CNS autoimmunity". J Autoimmun 84:1–11
Kim ML et al (2018) "Dysregulated IL-1beta-GM-CSF Axis in Acute Rheumatic Fever That Is Limited by Hydroxychloroquine. " Circulation 138(23):2648–2661
Kirk J et al (2003) 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 201(2):319–327
Kirk S et al (2004) "Angiogenesis in multiple sclerosis: is it good, bad or an epiphenomenon?". J Neurol Sci 217(2):125–130
Kirk SL, Karlik SJ (2003) "VEGF and vascular changes in chronic neuroinflammation". J Autoimmun 21(4):353–363
Koch MW et al (2021) "Hydroxychloroquine for Primary Progressive Multiple Sclerosis". Ann Neurol 90(6):940–948
Koch MW et al (2015) "Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis". J Neurol Sci 358(1–2):131–137
Lange C et al (2016) Vascular endothelial growth factor: a neurovascular target in neurological diseases. Nat Rev Neurol 12(8):439–454
Li Z et al (2020) "Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS". J Clin Invest 130(4):1977–1990
Liu XS et al (2009) "Angiopoietin 2 mediates the differentiation and migration of neural progenitor cells in the subventricular zone after stroke". J Biol Chem 284(34):22680–22689
Ma Y et al (2021) "The Role of Immune Cells in Post-Stroke Angiogenesis and Neuronal Remodeling: The Known and the Unknown. " Front Immunol 12:784098
Macmillan CJ et al (2011) Angiogenesis is regulated by angiopoietins during experimental autoimmune encephalomyelitis and is indirectly related to vascular permeability. J Neuropathol Exp Neurol 70(12):1107–1123
Miller DH, Leary SM (2007) "Primary-progressive multiple sclerosis". Lancet Neurol 6(10):903–912
Montalban X et al (2017) "Ocrelizumab versus Placebo in Primary Progressive Multiple Sclerosis". N Engl J Med 376(3):209–220
Monti L et al (2017) "Influence of Circulating Endothelin-1 and Asymmetric Dimethylarginine on Whole Brain Circulation Time in Multiple Sclerosis. " Biomark Insights 12:1177271917712514
Motl RW et al (2017) "Validity of the timed 25-foot walk as an ambulatory performance outcome measure for multiple sclerosis". Mult Scler 23(5):704–710
Openshaw H et al (2000) Multiple sclerosis flares associated with recombinant granulocyte colony-stimulating factor. Neurology 54(11):2147–2150
Pache M et al (2003) "Extraocular blood flow and endothelin-1 plasma levels in patients with multiple sclerosis". Eur Neurol 49(3):164–168
Proescholdt MA et al (1999) Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain. J Neuropathol Exp Neurol 58(6):613–627
Proescholdt MA et al (2002) Vascular endothelial growth factor is expressed in multiple sclerosis plaques and can induce inflammatory lesions in experimental allergic encephalomyelitis rats. J Neuropathol Exp Neurol 61(10):914–925
Rahman R et al (2016) "The effects of hydroxychloroquine on endothelial dysfunction". Pregnancy Hypertens 6(4):259–262
Rahman RA et al (2020) "Hydroxychloroquine Mitigates the Production of 8-Isoprostane and Improves Vascular Dysfunction: Implications for Treating Preeclampsia."Int J Mol Sci21(7)
Rasol HA et al (2016) "Vascular endothelial growth factor-A mRNA gene expression in clinical phases of multiple sclerosis". Ann Clin Biochem 53(Pt 2):252–258
Rezabakhsh A et al (2017) "Effect of hydroxychloroquine on oxidative/nitrosative status and angiogenesis in endothelial cells under high glucose condition. " Bioimpacts 7(4):219–226
Rocha NP et al (2019) "Exploring the relationship between Endothelin-1 and peripheral inflammation in multiple sclerosis. " J Neuroimmunol 326:45–48
Sarchielli P et al (2008) Fibroblast growth factor-2 levels are elevated in the cerebrospinal fluid of multiple sclerosis patients. Neurosci Lett 435(3):223–228
Schenk GJ et al (2013) Roles for HB-EGF and CD9 in multiple sclerosis. Glia 61(11):1890–1905
Schreiber K et al (2018) The effect of hydroxychloroquine on haemostasis, complement, inflammation and angiogenesis in patients with antiphospholipid antibodies. Rheumatology (Oxford) 57(1):120–124
Shin T et al (2001) "Intrathecal administration of endothelin-1 receptor antagonist ameliorates autoimmune encephalomyelitis in Lewis rats. " Neuroreport 12(7):1465–1468
Snir O et al (2006) "G-CSF enhances the adhesion of encephalitogenic T cells to extracellular matrix components: a possible mechanism for exacerbation of multiple sclerosis". J Neuroimmunol 172(1–2):145–155
Speciale L et al (2000) Endothelin and nitric oxide levels in cerebrospinal fluid of patients with multiple sclerosis. J Neurovirol 6(Suppl 2):S62–66
Toledo FGS et al (2021) "The effects of hydroxychloroquine on insulin sensitivity, insulin clearance and inflammation in insulin-resistant adults: A randomized trial". Diabetes Obes Metab 23(6):1252–1261
Tottenham I et al (2022) "Serum HGF and APN2 are associated with disability worsening in SPMS. " J Neuroimmunol 364:577803
Trapp BD, Stys PK (2009) "Virtual hypoxia and chronic necrosis of demyelinated axons in multiple sclerosis". Lancet Neurol 8(3):280–291
Ukkonen M et al (2007) "Cell surface adhesion molecules and cytokine profiles in primary progressive multiple sclerosis". Mult Scler 13(6):701–707
Yong HYF, Yong VW (2022) Mechanism-based criteria to improve therapeutic outcomes in progressive multiple sclerosis. Nat Rev Neurol 18(1):40–55

No competing interests reported.

Download PDF

Editorial decision: Major revision
17 Oct, 2023
Editorial decision: Revision requested
17 Oct, 2023
Reviews received at journal
10 Oct, 2023
Reviewers agreed at journal
27 Sep, 2023
Reviewers agreed at journal
20 May, 2023
Reviews received at journal
13 May, 2023
Reviewers agreed at journal
08 May, 2023
Reviewers agreed at journal
04 Apr, 2023
Reviewers invited by journal
01 Mar, 2023
Editor assigned by journal
03 Jan, 2023
Submission checks completed at journal
06 Oct, 2022
First submitted to journal
03 Oct, 2022

You are reading this latest preprint version

Hydroxychloroquine modifies angiogenesis factors in primary progressive multiple sclerosis

Status:

Version 1

Abstract

Figures

1.0 Introduction

2.0 Materials And Methods

2.1 Study Design and Participants

2.2 Study Ethics

2.3 Angiogenesis Markers

2.4 Outcomes

2.5 Analysis and Descriptive Statistics

3.0 Results

3.1 Patient Characteristics

3.2 Angiogenesis Markers at Baseline

3.3 The Effect of Hydroxychloroquine on Angiogenesis Markers Based on Longitudinal Dynamics

3.4 Angiogenesis Regulation by Hydroxychloroquine in PPMS appears to Correlate with Disability Progression

4.0 Discussion

4.1 Angiogenesis may play dichotomous roles in multiple sclerosis and other inflammatory disorders

4.2 The effect of hydroxychloroquine on angiogenic factors in multiple sclerosis and possible mechanism of action

4.3 Possible mechanisms for which hydroxychloroquine affects angiogenic factors

4.4 Limitations and Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1