Pseudo-Exfoliative Glaucoma: Our Experience

Abstract

We investigated the correlation between hyperhomocysteinemia and pseudo-exfoliative glaucoma. After providing extensive background information, we outlined our study methodology. We assembled a control group of 20 individuals, considering their medical history (including hypertension, diabetes, cardiovascular and cerebrovascular diseases, nephropathies, and inappropriate drug therapy). Our study focused exclusively on patients with secondary open-angle glaucoma associated with pseudo-exfoliation, which is the most common cause of open-angle glaucoma. Our finding indicates that hyperhomocysteinemia is significantly elevated in subjects with pseudo-exfoliative glaucoma compared to individuals without ocular pathology but with a similar vascular risk. Homocysteine, by promoting the overproduction of free radicals, damages the intima of blood vessel walls and triggers elastase release in arterial smooth muscle cells. Antioxidants play a crucial role in mitigating the harmful effects of hyperhomocysteinemia, and folic acid supplementation, either alone or in combination with vitamins B12 and B6, improves endothelial function.

1. Introduction

Pseudo-exfoliative glaucoma is the most common cause of secondary glaucoma. Pseudo-exfoliative syndrome, from which it takes its name, is the main risk factor for the onset of glaucomatous disease, the second most common cause of visual impairment and blindness in Italy [1].

We refer the interested reader to Topouzis et al. [2] for a discussion of risk factors for glaucoma in a specific population; in addition, interested readers will find a detailed discussion of pseudo-exfoliative glaucoma in two very recent papers Washington et al. [3] and Yuksle et al. [4].

Homocysteine is an amino acid that plays a major role among the predisposing factors for thrombophilia, and the development of systemic and vascular pathologies and its role in pseudo-exfoliative glaucoma has been illustrated in [3].

Our study showed that there was a correlation between patients with pseudo-exfoliative glaucoma and the actual presence of hyperhomocysteinemia.

2. Background

2.1 Intraocular pressure (IOP)

The balance between aqueous humour production and absorption in the anterior chamber is crucial for pressure which usually ranges between 10 and 20 mmHg. In the glaucoma patient, this mechanism fails, hindering the outflow of aqueous humour, which, as it accumulates, causes an increase in intraocular pressure (IOP). This increase in pressure will result in mechanical damage at the head of the optic papilla. The damage caused by the increased pressure will also have a microvascular effect on the optic nerve with relative hypoperfusion and apoptosis of its nerve fibres. The loss of all nerve fibres will irreversibly result in blindness.

2.2 Glaucoma

Glaucoma is a very large group of ocular neuropathies characterised by progressive and irreversible damage to the optic nerve Gelormini et al. [5] and Caporossi et al. [6] for a general introduction and Flammer [7] for a more focused survey on glaucoma. The acute onset rarely occurs, often the course is subtle and progressive, resulting in functional damage to the visual field. In the past, the Von Graefe triad, which required a high IOP above 21 mmHg, functional visual field defects, and papilla neuropathy were used to detect glaucoma Flammer [7].

A number of studies have shown that many glaucomas do not have a high IOP and just as many have visual field damage, but only detected late.

In the face of these findings, of the previous triad, the only remaining stronghold is optic neuropathy Flammer [7].

Thus, one can speak of glaucoma considering optic nerve damage. An elevated IOP does not indicate pathology, nor can the presence of glaucoma lead to an elevated IOP.

2.3 Classification of glaucoma

We can distinguish glaucoma into two groups:

1. Clinically evident glaucoma, a much rarer manifestation with more or less obvious symptomatology and simply diagnosable among manifest papillary damage and high IOP. In this group, we include acute glaucoma, congenital glaucoma, and secondary, albeit more subtle and often chronic glaucoma.

2. Clinically silent glaucoma, much more common than the former.

We can also classify glaucoma according to the correlation of another ocular pathology thus distinguishing:

1. Primary glaucoma when there is no concomitant second pathology.

2. Secondary glaucoma when it is accompanied by a different ocular pathology.

We can make use of a third, more commonly accepted classification, which is based on the pathophysiology of the aqueous humour and its outflow, recognising two different pathologies based on the chamber angle:

1. Open-angle glaucoma.

2. Angle-closure glaucoma.

There is no primary classification or one that is not taken into account, but they can intersect with each other. We can therefore have, for example, clinically evident, secondary, closed-angle glaucoma.

Open-angle glaucoma is the most common type of glaucoma in this category and is mainly influenced by genetic predisposition; risk factors such as diabetes or myopia can have a significant impact on the development of open-angle glaucoma Flammer [7]. Interactions of some genes with glaucoma have been discovered, among them the GLC 1A gene and the Duffy group, a blood marker, both on chromosome 1. Non-modifiable risk factors include older age and ethnicity. By the time an alteration of the visual field occurs, optic nerve atrophy is already advanced.

2.4 Pseudo-exfoliative glaucoma

Secondary open-angle glaucoma includes glaucoma due to pseudo-exfoliative syndrome (PEX syndrome). We refer the reader to Flammer, Kozart and Yanof, and Ritch [7, 8, 9] for an introduction on exfoliative syndrome. The association between exfoliation syndrome and glaucoma has been reviewed in Ritch [10] and Mitchel et al. [11]; we also refer the interested reader to Jeng et al. [12] for the results of a study carried out in a very specific population. More recently, some studies explored the genetic association between pseudo-exfoliative syndrome and glaucoma Schlötzer-Schrehardt [13], Thorleifsoon et al. [14] and Ye et al. [15]. In addition, Ritch [16] and Ritch [17] describe ties between exfoliation syndrome and open glaucoma; Jeng [12], Forsius [18], and Ringvold et al. [19] discuss empirical results obtained on some specific populations. However, we point out that the exfoliation syndrome has been associated with other type of diseases such as vascular diseases Praveen et al. [20], Mitchell et al. [21], Shrum et al. [22], Schumacher et al. [23], Alzheimer Linner et al. [24] and Cumurcu et al. [25], and to some extent, it can be regarded as a a systemic disorder [26, 27, 28].

PEX syndrome is a systemic extracellular matrix (ECM) disorder characterised by the production and gradual accumulation of fibrillar material, not only at the skin level, but also at the connective tissue level of various organs, leading to glaucoma and increased cerebrovascular and cardiovascular morbidity such as Alzheimer’s disease [24, 25], hearing loss [29, 30, 31], and increased plasma levels of homocysteine that increase the risk of thrombotic events.

PEX was first defined by Lindberg in 1917 and associated with glaucoma only a few years later in 1924.

It represents the most common form of secondary open-angle glaucoma with approximately 70 million people suffering from PEX and of these about 10% develop chronic open-angle glaucoma.

This term, pseudo-exfoliative, was chosen because clinically it has been observed that the lens appears to exfoliate but this is not what happens during this disease. Some important studies focus on the clinical detection of exfoliation syndrome [32, 33, 34, 35]; the variation of IOP in patients with exfoliation glaucoma and patients with primary open-angle glaucoma is extensively analysed in Konstas et al. [36], while Konstas et al. [37] discuss the factors influencing the progression of exfoliation glaucoma. Medical treatments for exfoliation glaucoma are presented in [38, 39, 40]; biomarkers for exfoliation glaucoma are reviewed in [36, 41]. The role of connective tissue growth in exfoliation glaucoma is investigated by Browne et al. [42]; also humour aqueous plays an important role, as emerges from [43, 44, 45, 46]. At the surgical level, some important studies are proposed in Shingleton et al. [47] and Yazgan et al. [48].

Since not all patients with PEX syndrome go on to develop glaucoma, a distinction must be made between PEX syndrome, PEX syndrome-associated glaucoma, and pseudo-exfoliative.

PEX syndrome presents grey/white deposits formed by an abnormal protein material found on any ocular surface wetted by aqueous humour. It is most easily seen on the anterior surface of the crystalline lens where it presents with a characteristic picture with a central disc and a peripheral band due to iris movements rubbing against the lens during mydriasis and miosis movements and enucleating parts of the deposited material.

This rubbing also generates a loss of pigment from the deep layers of the iris, which is highlighted on transillumination. The light that penetrates through the pupil is reflected by the fundus and usually exits through the iris, which by its structure acts as an optical diaphragm. This mechanism fails in areas where there has been a greater loss of pigment at the level of the iris.

The affected areas appear red due to the backlighting of the light reflection from the ocular fundus because they are highly vascularised to the point of calling the colour ‘fundus red’.

This phenomenon also explains the reason for red eyes in photos taken with flash.

At the level of the anterior capsule of the lens, these deposits of furfuraceous material take on an identifiable arrangement in three zones: a central disc, a peripheral granular zone, and an intermediate ring without deposits.

It is also important that pseudo-exfoliative material is found at the level of the corneal Descemet’s membrane, and instead at the level of the iris and trabecular meshwork where it causes an obstruction of aqueous humour outflow with a related increase in IOP, leading to the onset of pseudo-exfoliative glaucoma.

These deposits can cause other ocular pathologies, e.g. increased fragility of the zonular fibrils that hold the lens in place, leading to possible complications in cataract surgery.

PEX syndrome is an age-related condition, generally from the sixth decade of life and is more common than one would think and fibrillar material can also occur in other organs.

The fibrillar material appears as a bush and can be observed under light microscopy. The electron microscopic presentation of pseudo-exfoliative disease in ocular and extra-ocular tissues was demonstrated by Schlötzer-Schrehardt.

The aetiology of this disease is unknown but has been classically linked to an altered metabolism of elastin fibrils. This hypothesis was confirmed by a study that demonstrated polymorphisms in the gene for lysyl oxidase-like protein 1 (LOXL1) [15]. This enzyme is involved in elastin metabolism and confers an increased risk of developing PEX syndrome. Several variants were found related to the POMP, CACNA1A, and SEMA6A genes, all three of which are linked to extracellular matrix metabolism, the ubiquitin-proteasome system, calcium signalling, and lipid biosynthesis in the pathogenesis of pseudo-exfoliation, increasing the risk of disease.

It is common in Scandinavian and Mediterranean populations, African Bantus, and Australian Aborigines, and moreover, it is more common in females.

A distinction should be made between primary open-angle glaucoma and pseudo-exfoliative glaucoma: usually, in the pseudo-exfoliative type, the IOP increases very quickly with considerable fluctuations that may result in greater damage to the optic disc than a high IOP yes, but still stable. Glaucomatous damage is also represented by the vascular changes associated with PEX glaucoma.

Another alteration due to PEX syndrome is the reduction of tear secretion and tear film stability.

One study showed that the tear osmolarity in both eyes of patients with clinically unilateral PES is higher than in normal subjects.

Small deposits of subjects with pseudo-exfoliative disease deposit on the corneal endothelium pseudo-exfoliation cells with reduced density.

Damaged corneal endothelium can cause endothelial decompensation. Pleomorphism in pseudo-exfoliation keratopathy with glaucoma is more frequent than with cataracts.

Recent studies using optical coherence tomography angiography have demonstrated a decrease in peripapillary and macular vascular density in patients with pseudo-exfoliation, suggesting that the vascular component, including optic nerve hypoperfusion, may be involved in the aetiopathogenesis of pseudo-exfoliative glaucoma.

3. Homocysteine

Homocysteine is a non-protein amino acid produced by the metabolism of methionine, an essential sulphur amino acid that is introduced into the body via the protein in the diet. Homocysteine is formed from S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The transformation from SAM to SAH occurs via trans-methylation processes.

The SAM/SAH ratio underlies the regulation of methionine-homocysteine metabolism. In a well-functioning organism, homocysteine is converted back into methionine, or into simple amino acids, which are easily eliminated from the body via the urine. Approximately 80% of homocysteine in the blood is bound to proteins, mainly albumin, via a disulphide bond. The unbound portion of homocysteine is easily oxidised to form disulphides: due to the presence of its free SH group, it can combine with another homocysteine to form the homocysteine dimer or with cysteine to form the mixed cysteine-homocysteine disulphide. The set of these non-protein complexed forms (20% of total homocysteine) is called free homocysteine. This includes, therefore, both the reduced (SH) and the oxidised form: (S‒S).

The strongly endothelium-toxic reduced form (SH) is approximately 2% and increases when plasma total homocysteine exceeds 100 μmol/L.

3.1 Homocysteine remethylation

Homocysteine can be remethylated into methionine by two processes:

In the former, with the presence of folic acid, in the folate cycle, the key reaction takes place thanks to the enzyme methylene tetrahydrofolate reductase (MTHFR), the coenzyme of which is vitamin B2, which reduces 5,10-methylene-tetrahydrofolate to 5-methyltetrahydrofolate; the latter will then provide, again with vitamin B12, the methyl group necessary for the reconversion of homocysteine to methionine.

In the second process, the remethylation reaction is carried out by the enzyme betaine synthase, which produces methionine by catalysing the transfer of a methyl group to homocysteine.

3.2 Hyperhomocysteinemia

The test to analyse homocysteine values in the blood is called homocysteinemia and consists of a venous blood sample.

Before the examination, it is preferable to fast for 10–12 hours and avoid smoking for at least 15 minutes prior to taking the sample.

Plasma homocysteine values are considered normal when they are around 5–12 μmol/L.

Hyperhomocysteinemia occurs when values exceed the maximum cut-off.

Let us distinguish hyperhomocysteinemia:

• Mild when the plasma value remains in the range of 12–15 μmol/L;

• Moderate when the range is 15–30 μmol/L;

• intermediate when between 30 and 100 μmol/L;

• Severe hyperhomocysteinemia when it exceeds the maximum cut-off.

The plasma concentration of homocysteine is the result of a close relationship between dietary habits and predisposing genetic factors.

Most people have high levels of homocysteine in their blood due to a diet that is not sufficiently rich in folic acid and the other B vitamins.

Other causes of hyperhomocysteinemia are genetic alterations that cause deficits in the enzymes involved in the metabolic cascade whose intermediate product is homocysteine.

In addition to deficiency of the enzyme cystathionine-beta-synthetase, due to a very rare genetic mutation, homocystinuria, elevated homocysteine levels may also be due to mutation of the gene responsible to produce the enzyme methylenetetrahydrofolate reductase (MTHFR). A genetic polymorphism has been identified as responsible for the increased homocysteine levels, characterised by the 1298A/C and C677T mutations; with the latter being more important in terms of thrombotic risk, resulting in a 50% reduction in MTHFR enzymatic activitỳ.

Genetic causes outnumber dietary/behavioural ones 1:10.

3.3 Risks of hyperhomocysteinemia

The patient with hyperhomocysteinemia is an individual with a high predisposition to thrombophilia and thus to cardiovascular disease.

Homocysteine has been shown to influence vascular function through indirect action on muscle tone by inducing increased vascular constriction mediated by the binding of reduced homocysteine with nitric oxide and related nitrous oxide formation [49, 50]. Several researchers explored the role of homocysteine and acute coronary syndromes [50], abdominal aortic aneurysm [51], atherosclerosis [52], retinal vein occlusion [53], diabetic retinopathy [54, 55, 56], and ischemic strokes [57, 58]. Other important studies focus, instead, on the impact of some drugs and foods on homocysteine levels [59, 60, 61, 62, 63, 64].

Chronically elevated homocysteine levels result in nitric oxide depletion, with nitrous oxide production remaining in the circulation for only 14 minutes. The resulting consequence is the patient in continuous vasospasm. A direct influence of high serum homocysteine levels causes atherosclerotic plaque formation and smooth muscle cell proliferation due to endothelial damage and reduced elasticitỳ. This is due to excess homocysteine forming the homocysteine-thiolactone complex, which reacts with LDL to generate an insoluble LDL-thiolactone complex. This complex is phagocytosed by macrophages which, unable to metabolise it, turn into foamy cells acting as an atheromatous core.

Excess homocysteine can also act as a free oxygen radical causing first endothelial dysfunction and then necrosis of the endothelial cells themselves with their subsequent detachment from the vessel wall. There may also be a proliferation of smooth muscle cells resulting in fibrocalcification of the vessel wall and oxidation of membrane lipids with loss of function of these structures.

Again, related to increased thrombotic risk, excessive homocysteine is also a strong platelet aggregator.

3.4 Therapy

Therapy is based first and foremost on a correction of the patient’s lifestyle by making changes to the diet.

In fact, the body can decrease serum homocysteine levels by means of folates and B vitamins, which are crucial in its metabolism.

It is essential for the patient to consume more food with folic acid and vitamin B12: the recommended intake levels for folic acid are 200–1000 mcg/day.

4. Correlation between pseudo-exfoliative glaucoma and hyperhomocysteinemia

4.1 Markers

Corneal biomechanical properties of subjects with pseudo-exfoliative syndrome, including corneal hysteresis, corneal resistance factor, and central corneal thickness, were reported to be decreased compared to healthy control subjects.

These changes were more pronounced in patients with pseudo-exfoliative glaucoma than in patients with pseudo-exfoliative syndrome.

Two interesting systemic laboratory markers for predicting the risk of progression from pseudo-exfoliative syndrome to pseudo-exfoliative glaucoma are the neutrophil to lymphocyte ratio and the platelet to lymphocyte ratio. Indeed, signs of subclinical systemic inflammation, including neutropenia and lymphocytopenia, became more pronounced in patients with PEX syndrome and even more so in those with pseudo-exfoliative glaucoma than in normal healthy subjects.

This finding suggests that inflammation plays a key role from the onset of PEX syndrome and can be used as a marker at follow-up to predict progression to pseudo-exfoliative glaucoma.

Chronic subclinical inflammation was also demonstrated locally in the anterior chamber.

Levels of activation-derived complement components were significantly elevated in the aqueous humour of patients with pseudo-exfoliative glaucoma compared to non-syndromic PEX glaucoma controls.

Complement inhibitors, including clusterin and vitronectin, were significantly elevated.

The association of clusterin with exfoliation fibrils suggests an unsuccessful attempt by this chaperonin to prevent the accumulation of fibrillar material. Apolipoprotein D (ApoD) is a secreted glycoprotein with multiple functions, including the prevention of lipid peroxidation. ApoD expression is upregulated during ageing and in several pathological conditions, including atherosclerosis, neurological diseases, and several types of cancer. Several studies have suggested that ApoD plays a protective role against oxidative stimuli.

This role was found to be diminished in PEX syndrome, where lower levels of this biomarker were detected in the aqueous humour of patients with pseudo-exfoliative syndrome.

Endothelin-1 is a peptide produced by the vascular endothelium with potent vasoconstrictor properties.

It appears to be involved in the regulation of ocular blood flow. High levels of endothelin-1 have been reported in the aqueous humour of patients with PEX syndrome. High levels of homocysteine have been found in both aqueous humour and tears.

High levels of this non-protein amino acid contribute to the pathogenesis of pseudo-exfoliative disease.

4.2 Purpose of the study

Both PEX and hyperhomocysteinemia are risk factors for systemic vascular and ocular disease.

We studied the correlation of hyperhomocysteinemia in patients with pseudo-exfoliative glaucoma compared to a healthy control group.

4.3 Methods

For each patient, we considered her/his medical history (hypertension, diabetes, cardiovascular and cerebrovascular diseases, nephropathies, and inappropriate drug therapy).

The control group was formed by 20 individuals without the disease who underwent a complete eye examination, including fundus and optic disc examination, visual field analysis, and other routine examinations to detect any elements that might affect the study.

We only included patients with secondary open-angle glaucoma with pseudo-exfoliation, the most common cause of open-angle glaucoma.

The study group consisted of glaucomatous patients diagnosed with pseudo-exfoliative glaucoma if clinical examination revealed fibrillar deposits on the anterior lens capsule and at the level of the ciliary angle with associated peripupillary atrophy and transillumination of the pupillary margin, elevated intraocular pressure, incipient optic nerve atrophy, and glaucomatous visual field defects.

Patients were excluded if they did NOT have a diagnosis of pseudo-exfoliative glaucoma based on clinical examination and visual field analysis, if they had diseases that could be associated with hyperhomocysteinemia, such as gastrointestinal malabsorption or diabetes mellitus, if they were abusing drugs and/or alcohol, and if they were receiving any pharmacological treatment, including methotrexate therapy and other possible vitamin supplements, in the 6 months prior to the study. Patients with glaucoma and on medical therapy were not excluded, as no glaucoma therapy is known to alter serum homocysteine levels.

For each participant in both the control and study groups, a 2 ml venous blood sample was collected and centrifuged for 6 minutes using the fluorescence-based high-performance liquid chromatography method.

A plasma value above 16 μmol/L is considered high.

The control group consisted of the same number of healthy adults, 20, again divided into 10 men and 10 women, with a mean age close to that of the study group, ranging from 64 to 76 years (mean 70 ± 6.1).

It was immediately apparent that the mean plasma homocysteine level was significantly higher in the pseudo-exfoliative glaucoma group.

Fifty percent of the patients in the pseudo-exfoliative glaucoma group had plasma levels to report hyperhomocysteinemia; in the control group the number was drastically lower: only 2/20 (10%).

Various cardiovascular diseases were found in the study group. For example, four patients (20%) had systemic hypertension, and two (10%) had ischaemic heart disease.

The two patients with pseudo-exfoliative glaucoma and ischaemic heart disease had elevated plasma homocysteine levels, as did 3 of the 4 patients with systemic hypertension.

In the control group, cardiovascular disease was found in four patients with systemic hypertension and one patient with ischaemic heart disease.

The patient with ischaemic heart disease and one of the four patients with hypertension had hyperhomocysteinemia (Table 1).

	No. of subjects	Age (in years)	Homocysteinemia	Hyperhomo-cysteinemia (%)
Study group	20	65–75; 70 ± 5.2	16.6 ± 3.1	50
Control group	20	64–76; 70 ± 6.1	12.41 ± 1.8	10

Table 1.

Features of the patients involved in our study.

5. Discussion

We found that the prevalence of hyperhomocysteinemia is significantly increased in subjects with pseudo-exfoliative glaucoma compared to subjects without ocular pathology but with a similar vascular risk.

Homocysteine can induce overproduction of free radicals which, by causing damage to the intima of the vessel wall, trigger elastase at the level of arterial smooth muscle cells.

This activation leads to elastolysis of elastin and fibrillar collagen in the arteries through activation of extracellular matrix metalloprotease and may explain the possible effect of homocysteine in systemic cardiovascular disease.

Antioxidants such as vitamin E and ascorbic acid are able to generate a significant reduction in the harmful effects of hyperhomocysteinemia.

Treatment supported by folic acid, which is able to eliminate free oxygen radicals, led to a general improvement in endothelial function, alone or in combination with the intake of B vitamins (B12 and B6).

6. Conclusions

The pseudo-exfoliative material contains both elastin and fibrin, and homocysteine is able to activate an elastase inducing elastolysis of elastin and fibrillar collagen.

This correlation could highlight new developments to better understand how fibrillar material is produced in pseudo-exfoliative disease and consequently take a step forward in the screening of pseudo-exfoliative glaucoma and the plausible role homocysteine plays in the pathogenesis of vascular disease in both the systemic and ocular spheres.

Indeed, there is a possibility, yet to be confirmed, that the pseudo-exfoliative material generated is actually a by-product of homocysteine-induced elastolysis.

A further line of development could be to evaluate the role of folic acid in pseudo-exfoliative glaucoma patients with hyperhomocysteinemia on IOP.