Predictive molecular profiling in blood of healthy vasospastic individuals: clue to targeted prevention as personalised medicine to effective costs.

Paradigm change from late interventional approach to predictive diagnostics followed by targeted prevention before manifest pathology, presents innovative concept for advanced healthcare. Preselection of healthy but pathology-predisposed individuals is the primary task in the overall action. Vasospasm is a frequent syndrome defined as an inappropriate constriction or insufficient dilatation in microcirculation. Vasospastic individuals are considered as healthy subpopulation predisposed to several pathologies including neurodegeneration. Clinical observations, subcellular imaging and "gene hunting"-investigations provide evidence for vasospasm as predisposition to glaucoma; development of further related pathologies cannot be excluded. Predictive molecular-profiling in blood can specify individual predisposition for effective prevention.

Vasospastic Syndrome: definition, prevalence, detection

Vasospastic Syndrome or vascular deregulation (VD) is defined as an inappropriate constriction or insufficient dilatation in the microcirculation (see Fig. 1).

Fig. 1

Alterations in conjunctival vessels in patients with vasospasm [1]

Generally, vasospasm is considered as primary and secondary one. Secondary VD is due to other diseases such as autoimmune one. Primary VD is prevalent in younger subpopulation, can potentially predispose to several disorders being, therefore, particularly attractive for predictive diagnostics and individualised treatment. Primary VD demonstrates following particularities:

it occurs more frequent in females manifested in puberty and moderating with age

this phenomenon is even more frequent in Japanese population compared to Caucasian [2]

usually academics are more affected by VD [3]

to clinical signs belong an inborn increased sensitivity to any kind of stress provocation (mechanical, cold, emotion, etc.), altered drug sensitivity, frequently cold extremities, altered sleep behaviour, reduced feeling of thirst, low blood-pressure, reduced body-mass-index, more frequent migraine compared to general population [1, 4, 5]

Compared to general population, vasospastic individuals tend to a meticulous personality and successful professional career [6].

A valuable diagnostic tool for the ascertainment of vasospastic diathesis is the nailfold capillary microscopy (see Fig. 2).

Fig. 2

Nailfold capillary microscopy (right) enables the monitoring of microcirculation and cold provocation of vasospasm in fingers (left) [1]

The best known blood-related risk factor is an increased plasma level of endothelin-1 [1]. Since Vasospastic Syndrome is a frequent phenomenon in young subpopulations, this makes the task of prediction and targeted prevention of “down-stream” related pathologies particularly attractive from several points of view including economical aspects.

What is the impact of vascular deregulation in glaucoma pathology?

A wealth of literature points to the importance of haemodynamics in glaucoma pathology. Vasospasm is frequently observed in glaucoma patients [1, 7]. Ocular ischemia resulting from blood-flow deficits may play a major role in the initiation of glaucoma: hypoxia, followed by high secretion of excitatory amino acids and elevated levels of intracellular calcium results in the process of retinal ganglion cell death [8-10]. In our previous studies, we have demonstrated stable alterations in gene expression of circulating leucocytes isolated from glaucoma patients compared to healthy controls [11-14]. Further, significant similarities in expression profiles of circulating leucocytes between vasospastic individuals and glaucoma patients have been recently published [15]. However, the same publication reports also significant dissimilarities of molecular patterns as compared to both glaucoma patients and healthy controls; consequently, a development of both degenerative and non-degenerative pathologies different from glaucomatous optic nerve degeneration but related to primary vasospasm cannot be excluded in vasospastic individuals.

What is the potential impact of prediction and prevention of glaucoma in healthy vasospastic individuals?

Worldwide, 67 million patients are affected by the neurodegenerative eye disease glaucoma. Glaucomatous optic neuropathy (GON) is the second leading cause of permanent vision loss. GON is a chronic degenerative process, the onset of which is not possible to monitor by currently existing diagnostic tools. Early treatment has been reported to be highly beneficial for well-timed treatment measures to slow-down the disease progression [16]. As review in this journal-issue [17], molecular pathomechanisms of glaucoma demonstrate both a considerable overlap and remarkable particularities to some other neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Thus versus controls, the neuronal thread protein (NTP) demonstrates enhanced expression levels in glaucoma, patients with Down Syndrome, Alzheimer’s and some other neurodegenerative diseases indicating the axonal lesions. However, whereas the accumulation of TAU-protein is characteristic for Alzheimer’s disease and other tauopathies, glaucoma patients do not demonstrate an increase in the target protein versus controls [16, 18]. Therefore, a potential predisposition of vasospastic individuals to related pathologies should be thoroughly examined. In this context, molecular pathways involved in vasospastic deregulation should be investigated from viewpoint of

identification of possible similarities as well as dissimilarities in molecular pathways between healthy vasospastic individuals and potential related pathologies developed later in life

specificity for predictive diagnostics of glaucoma pathology in predisposed vasospastic individuals should be strictly validated against several control groups including other neuro/degenerative diseases

selection of molecular targets should be performed for vasospastic individuals in favour of non-invasive (blood test) diagnostic approaches followed by personalised treatment towards individual predisposition to single pathologies.

A monitoring of the pathology-specific molecular patterns is particularly valuable to develop reliable diagnostic approaches before the manifest pathology. Predictive tests can specify individual predisposition for well-timed preventive measures.

Similarities in subcellular images of DNA-damage and -repair capacity in vasospasm and glaucoma

Research work focused on the ex vivo comparative investigations of DNA damage in circulated leucocytes (CL) isolated from patients with glaucoma demonstrated significantly enhanced DNA damage compared to both healthy vasospastic and non-vasospastic individuals [19]. Comparative “Comet Assay” analysis revealed patterns of comets typical for glaucoma patients as shown in Fig. 3.

Fig. 3

Ex vivo “Comet Assay”-analysis of DNA damage in circulating leucocytes of glaucoma patients and non-glaucomatous vasospastic individuals versus healthy controls [19]. Figures A–D give examples of images typical for a. control group, b. group of vasospastic individuals, c. group of normal-tension glaucoma patients, and d. group of high-tension glaucoma patients. Comet-patterns typical for healthy controls (A) show that chromosomal DNA is localised mainly to heads of comets (intact DNA). In contrast, images B, C, and D demonstrate clearly damaged DNA (visible comet-tails and diffuse comet-heads). Evaluation of the shape of a comet allows for assessment of DNA damage. Individual migration patterns are demonstrated in groups corresponding to the images A–D by the evaluation of comet-classes (class number progression corresponds to increasing damage degree) as described earlier [20]. Figure E shows mean values calculated for relative DNA damage in following groups as compared to the control group (100%): VI—vasospastic individuals (102 ± 7%), NTG normal-tension glaucoma (160 ± 13%), and HTG high-tension glaucoma (128 ± 10%) [18]

Although DNA damage in the vasospastic non-glaucomatous group is not found to be significantly increased versus healthy controls, DNA from vasospastic individuals showed highly group-specific comet-patterns with the degree of damage intermediate between healthy controls and glaucoma patients. These findings indicate “comet assay” profiling of DNA-damage in CL as a potentially powerful tool for the non-invasive early/predictive molecular diagnostics of glaucoma disease in vasospastic individuals [18]. Furthermore, unrepaired DNA-damage in vasospastic individuals can lead to several pathologies different from glaucomatous optic nerve degeneration. This predisposition should be thoroughly investigated and the specificity of “Comet Assay”-patterns of vasospastic individuals should be validated comparing with patterns of other degenerative and non-degenerative pathologies. Thus, “Comet Assay”-analysis as a suitable tool for biomarkers has also been suggested for another neurodegenerative disorder—Alzheimer’s disease [21]. “Comet Assay”-analysis reveals enhanced DNA damage in both high- and normal-tension glaucoma [19]. Whether the level of DNA-damage correlates with disease severity, or not remains currently unclear. Further studies should also evaluate, whether a significant increase in DNA damage of leucocytes of glaucoma patients is caused by either disease specific stress factors, such as local ischemic/reperfusion events, and/or decreased capacity of DNA-repair machinery. There is some evidence for both eventualities: simultaneous up-regulation of p53 (stress regulated gene) and down-regulation of XPGC (essential member of DNA-repair machinery) have been ex vivo demonstrated in CL of glaucoma patients [22] and represent potential molecular blood markers for the disease.

Similarities in expression patterns detected in circulating leucocytes of vasospastic individuals and glaucoma patients

2D-PAGE

Protein-patterns in circulating leucocytes demonstrate clear similarities between vasospasm and normal-tension glaucoma versus controls. Moreover, protein-clusters can be considered for predictive imaging of healthy vasospastic individuals to glaucoma as shown in Fig. 4.

Fig. 4

Proteomics-imaging of blood-biomarkers (ex vivo identification in circulating leucocytes) specific for normal-tension glaucoma (NTG). A. Top: The pathology-specific protein-cluster is completely suppressed in both NTG and vasospasm in contrast to controls. B. Bottom: The marked protein SSP2602 is highly up-regulated in both NTG and vasospasm; this protein normally is not expressed by circulating leucocytes of controls—healthy individuals without vasospasm [22]

Expression array

The image of hybridised “AtlasTM Human Cardiovascular Array” revealed similarities as well as alterations in expression-patterns among normal-tension glaucoma (NTG), high-tension glaucoma (HTG), and VD groups versus controls are summarised in Table 1.

Table 1

Numbers of genes an expression of which is either differential or equal among the groups tested as shown by “Expression array”. Thereby, 108 genes were found to be differentially expressed between NTG and HTG groups. 34 genes demonstrated similar alteration for vasospastic individuals (VD) and both glaucoma-patient groups when compared to the healthy controls (see these genes listed in Table 2)

Differential to control	VD equal to	VD Differential to
NTG → 146	Control → 528	NTG → 109
HTG → 68	NTG → 48	K → 60
VD → 60	HTG → 43	HTG → 43
NTG = HTG → 53		NTG = HTG → 21

Table 2

Differentially expressed genes (altogether 34 ones as also summarised in Table 1) versus controls, the transcriptional levels of which were similar for VD, NTG and HTG groups [18]

Double-spot position in “EA”-image exp. difference vs. control	Name of gene as given in “AtlasTM Human Cardiovascular Array”	GenBank Accession	SwissProt Accession	Gene/Protein Classification
A7d	P2Y purinoceptor 7 (P2Y7); leukotriene B4 receptor; Chemoattractant receptor-like1 (CMKRL1)	U41070	Q15722	Other receptors (by Ligands)
increased			Q13305	G Protein-Coupled Receptors
			Q92641
A7e	Retinoic acid (Vitamin-A1-Säure) receptor gamma 1 (RAR-gamma 1; RARG)	M24857	P13631	Transcription Activator & Repressors
increased		M38258		Hormone Receptors
		M57707		Nuclear Receptors
		M32074
B1n	Androgen receptor coactivator 70-kDa subunit (ARA70)	L49399	Q13772	Transcription Activator & Repressors
increased
B4c	G protein-activated inward potassium channel 4 (GIRK4); heart K+/ATP channel (KATP1); cardiac inward rectifyer (CIR); KIR3.4	U39195	P48544	Voltage-gated Ion Channels
increased			Q92807
B4d	Sodium/calcium exchanger 1 precursor; Na+/Ca2+-exchange protein 1	M91368	P32418	Symporters & Antiporters
increased
B4e	Multidrug resistance protein 3 (MDR3); P-Glycoprotein 3 (PGY3)	M23234	P21439	Drug-Resistance proteins
increased				Xenobic Transporters
				ABC transporters
B5f	Endothelial nitric oxide synthase (EC-NOS)	M93718	P29474	Other Metabolism Enzymes
increased				Other Intracellular Transducers, Effectors & Modulators
B6d	Intercellular adhesion molecule 1 precursor (ICAM1); major group rhinovirus receptor; CD54 antigen	J03132	P05362	Matrix Adhesion Receptors
increased
B7g	Calcium & integrin-binding protein (CIB)	U85611	Q99828	Calcium-Binding proteins
increased
C1g	Cadherin 7 (CDH7)	AF047826	O60574	Cell Surface Antigens
increased				Cell-Cell Adhesion Receptors
C1h	Intestinal peptide-associated transporter 1 (HPT1)	U07969	Q12864	Other Cell Adhesion proteins
increased				Other Cell Adhesion Proteins
				Other Facilitated Diffusion proteins
C2i	GAP junction alpha-5 protein	L34954	P36382	Cell-Cell Adhesion Receptors
increased				Other Membrane Channels & Transporters
C3 m	Integrin beta 2 (ITGB2); cell surface adhesion glycoprotiens LFA-1/CR3/p150, 95 beta subunit precursor; CD18 antigen;	M15395	P05107	Cell-Cell Adhesion Receptors
increased	Complement receptor C3 beta subunit		Q16418
D1 m	Cardiac LIM domain protein; muscle LIM protein; cystein-rich protein 3 (CRP3); LIM-only protein 4	U49837	P50461	Basic Transcription Factors
increased				Other Transcription Proteins
				DNA Synthesis, Recombination & Repair Proteins
D1n	Cardiotrophin-1 (CT1)	U43030	Q16619	Growth Factors, cytokines & Chemokines
increased
D2n	Matrix metalloproteinase 16 (MMP-16)	D83646	P51512	Chromatin Proteins
increased				Metalloproteinases
D4a	TIMP-3	U14394	P35625	Extracellular Matrix Proteins
increased				Proteinase Inhibitor
D4b	TIMP-4	U76456	Q99727	Extracellular Matrix Proteins
increased				Proteinase Inhibitor
D4d	Sterol regulatory element-binding transcription factor 1	U00968	P36956	Basic trnascription Factors
increased				Other Apoptosis-Associated Proteins
D4e	Sterol regulatory element-binding transcription factor 2	U02031	Q12772	Basic trnascription Factors
increased				Other Apoptosis-Associated Proteins
D5 h	Rab geranylgeranyl transferase bety subunit	Y08201	P53611	Trafficking & Targeting Proteins
increased			Q92697	Protein Modification Enzymes
				GTP/GDP Exchangers & GTPase Activity Modulators
D6 m	Muscle-specific DNase I-like precursor (DNase 1 L1; DNL 1 L); Dnase X	X90392	P49184	DNA Synthesis, Recombination & Repair Proteins
decreased		L40817		Apoptosis-Associated Proteins
		U06846
E1b	Lanosterol synthase (LSS); oxidosqualene lanosterol cyclase (OSC)	D63807	P48449	Complex Lipid Metabolism
increased
E3n	NADPH-cytochrome p450 reductase	S90469	Q16455	Xenobic Metabolism
increased			P16435
E4 g	Steroid 5 alpha reductase 1 (SRD5A1); 3-oxo-5-alpha steroid 4 dehydrogenase 1	M32313	P18405	Complex Lipid Metabolism
increased		M68886
E4 h	Steroid 5-alpha reductase 2 (SRD5A2); 3-oxo-5-alpha steroid 4 dehydrogenase 2	M74047	P31213	Complex Lipid Metabolism
increased
F2d	Pregnane X receptor (PXR)	AF061056	O75469	Hormone receptors
increased				Nuclear Receptors
F2e	Estrogen-related receptor gamma	AF058291	O75454	Hormone Receptors
increased				Nuclear Receptors
F2f	Nuclear receptor subfamily 4 group A member 2 (NR4A2); nuclear receptor-related protein 1 (NURR1); transcriptionally inducuble nuclear receptor (TINUR); NOT	X75918	P43354	Hormone Receptors
increased				Nuclear Receptors
				Transcription Activators & Repressors
F2i	Orphan nuclear receptor TR4; nuclear receptor subfamily 2 group c member 2 (NR2C2); TAK1	U10990	P55092	Orphan Receptors
increased			P49116	Nuclear Receptors
				Transcription Activators & Repressors
F3a	RAR-related orphan receptor C	U16997	P51449	Orphan Receptors
increased				Nuclear Receptors
				Transcription Activators & Repressors
F3e	LX receptor alpha (LXR alpha)	U22662	Q13133	Orphan Receptors
increased
F3i	Platelet-activating factor acetylhydrolase IB alpha subunit	L13387	P43034	Other Metabolism Enzymes
increased
F7c	Myocyte-specific enhancer factor 2A (MEF2A); serum response factor-like protein 1	X68505	Q02078	Basic Transcription Factors
increased			Q14223
			Q14224

Compared to the control group the expression of 146, 68, and 60 genes were found to be altered in NTG, HTG, and VD groups respectively; the same 53 genes were differentially expressed in both NTG and HTG groups versus controls. Among 146 genes differentially expressed specifically in NTG group we monitored 48 and 53 genes which were similarly expressed either in VD or in HTG groups respectively. Among 68 genes differentially expressed specifically in HTG group we found 43 genes to be similarly expressed in VD group only. The highest difference—146 genes—was found to be between NTG and control groups. In contrast, the lowest difference—21 genes—was demonstrated to be between VD and the overlap of NTG/HTG.

34 genes demonstrated similar expressional alterations in NTG, HTG, and VD groups versus controls as given in Table 2. As the differentially expressed overlap VD/NTG/HTG was compared with the control group, following most significant difference was monitored:

P2Y purinoreceptor 7

Na+/Ca2+ exchange protein 1 (Na+/Ca2+ EP1)

Intercellular adhesion molecule 1 (ICAM1)

The cluster of the tissue remodelling metalloproteinases.

This group of gene-transcripts is proposed to be the reliable target to design advanced diagnostic tools for predictive glaucoma diagnosis in healthy vasospastic individuals [15].

P2Y purinoreceptor is upregulated in vasospastic individuals and glaucoma patients

The movement of leucocytes from blood into tissue is regulated by local production of chemo-attractants—diverse molecules, the chemotactic signal of which is transmitted by G-protein-coupled purinoceptor family P2Y. These receptors respond to chemotactic signals of traumatic, infectious, post-ischemic, autoimmune, and various toxic injuries. Extracellular nucleotides released from the activated platelets and other damaged cell types exacerbate the inflammatory response by cell specific leukotrene generation [23]. Thus, neutrophils generate leukotrienes B4 (LTB4) which are involved in the genesis of inflammation and oedema because of their effect on vascular permeability, plasma extravasation, diapedesis of white blood cells, and their important role in adaptive immune responses as reviewed by Di Gennaro et al. [24]. Specifically, a highly enhanced concentration of leukotrienes B4 and C4 has been observed in CSF of patients with multiple sclerosis [25]. The member of leukotrienes receptors family—LTB4 receptor or P2Y purinoceptor 7—for the first time has been isolated from human erythroleukaemia cell cDNA library [26]. The stimulation of monocytes, neutrophils, and endothelial cells was suggested to be a physiological role for the LTB4 receptor [27]. There is a growing body of evidence indicating an important role of LTB4 receptors in regulation of pathologic inflammation. Particularly using animal inflammatory models a reduced disease severity has been shown as LTB4 receptor antagonists have been applied; the same effect has been observed in mice with target deletion of BLT1—a high-affinity LTB4 receptor primarily expressed in leucocytes [28]. Furthermore, some studies support a potential role of P2Y receptors in controlling intraocular pressure, although additional investigations of the issue are necessary [29].

ICAM-1 is upregulated in vasospastic individuals and glaucoma patients

Neutrophil-endothelium interactions are implicated in pathological alterations of blood vessel function, potentially leading to circulatory disturbances [30]. Interactions between blood cells and the vessel wall result in endothelial dysfunction and injury leading to increased blood-brain barrier permeability and even oedema formation [31]. Penetration of leucocyte into inflamed areas involves complex interaction of leucocytes with endothelium through regulated expression of surface adhesion molecules. Found in this work to be highly expressed in VD, NTG and HTG groups ICAM-1 is believed to be largely responsible for the adhesion and trans-endothelial migration of leucocytes [32]. This is well in agreement with earlier developed strategies aimed at inhibition of endothelial interactions with leucocytes via use of adhesion molecule monoclonal antibodies, which successfully reduce cerebral ischemia/reperfusion injury, infarct size, and demonstrate a neuroprotective effect generally [33-35]. In our study, highly expressed ICAM-1 was found in leucocytes of glaucoma patients; in contrast, if any only traces of the target expression was detected in the leucocytes of healthy controls.

Sodium calcium exchanger (NCE)

Many studies examined the levels of cytosolic Ca2+ ([Ca2+]c) and Na+ ([Na+]c) in human blood cells, whereby leucocytes have been the main target of studying the relationship between blood pressure and intracellular content of both ions as reviewed by Horiguchi et al. [36]. As it has been shown by Horiguchi et al., the resting [Ca2+]c correlates well with NCE expression indicating NCE expression regulation to be an adaptive mechanism for Ca2+ extrusion mediation. The same study observed also a gender effect on [Ca2+]c / [Na+]c regulation in circulating leucocytes being in relationship with blood pressure. Further, the role of endothelial intracellular Ca2+ concentration in molecular mechanisms of vasoconstriction/vasodilatation has been intensively studied, and the functional association between P2Y purinoceptors, endothelial NO synthesis and calcium transport in terms of vascular regulation is well documented in the literature [37, 38]. Our findings here clearly demonstrate the up-regulation of both P2Y purinoceptor and Na+/Ca2+ exchanger in circulating leucocytes of glaucoma patients as well as vasospastic individuals versus healthy controls.

Tissue remodelling metalloproteinases

Significantly increased protein expression rates of both latent and active forms of metalloproteinases MMP-9 and MT1-MMP in circulating leucocytes correlate well with the enhanced levels of transcription and with glaucoma diagnosis [12]. Once activated, both hydrolases necessarily contribute to remodelling or even degeneration of the tissue whereto they are secreted by circulating leucocytes. This up-regulation might be a consequence of repeated mild ischemia/reperfusion postulated for both vasospastic individuals and glaucoma patients [18]. However, the question as to whether or not there is a correlation between an increased MMPs activity and glaucoma severity should be further clarified.

Furthermore, the increased synthesis of tissue-remodelling hydrolases detected in blood of healthy vasospastic individuals can potentially lead to development of some other pathologies which have not been considered as related to vasospasm till now. To the potential list of them belong altered wound-healing, some types of organ-degeneration, increased metastases activity. Large-scale studies are essential to be preformed, in order to prove a potential impact of vasospasm for the above listed pathophysiologic processes / manifested pathologies. This allows a targeted prevention at the stage of pre-lesions, “upstream” disease manifestation.

Concluding remarks

Expression similarities between glaucoma and VD versus controls indicate, on one side, a predisposition of VD individuals to glaucomatous damage, and, on the other side, an important role of vascular component in glaucoma pathology.

Expression dissimilarities between VD and glaucoma patients might indicate some glaucoma-specific pathomechanisms which are not involved in the stage of predisposition by VD.

Both expression similarities and dissimilarities could be useful to ascertain predictive diagnosis of glaucoma.

Molecular rearrangement in leucocytes of both VD and glaucoma patients has been shown to be typical for circulating leucocytes during vascular injury as reviewed by Kunapoli and Daniel [39] and includes

➢ an up-regulated adhesive protein expression via ICAM1

➢ an induced chemo-taxis via P2Y purinoceptors

➢ a mobilisation of intracellular Ca2+ response via Na+/Ca2+ exchanger, and

➢ the core of tissue remodelling metalloproteinases.

Pathology-specific molecular and subcellular patterns may create the basis for the development of more specific and non-invasive molecular imaging technologies in early/predictive glaucoma diagnostics.

Development of some other “down-stream” pathologies different from glaucoma but related to primary vasospastic syndrome cannot be excluded.

Predictive molecular-profiling in blood can specify individual predisposition for effective prevention to lower costs compared to those for treatment of manifest pathologies.