Helmut Schatz

Vitreous levels of the insulin-like growth factors I and II, and the insulin-like growth factor binding proteins 2 and 3, increase in neovascular eye disease. Studies in nondiabetic and diabetic subjects.

Retinal capillary nonperfusion results in neovascularization of the eye, which is restricted to the retina in less severe cases and progresses to the anterior chamber and the iris angle in the most advanced case, called rubeosis. This angioneogenesis may be induced by the release of retinal growth factors into the vitreous. This study compared levels of the IGF-I and IGF-II, and of the IGF binding protein-2 (IGFBP-2) and IGFBP-3 in vitreous from three groups with different degrees of retinal ischemia, as judged by the extent of neovascularization: a control group without new vessel formation, retinal neovascularization in patients with proliferative diabetic retinopathy, and massive ischemia of various causes resulting in rubeosis. IGF-I and IGFBP-3 were increased 10- and 13-fold in rubeosis (P << 0.01) compared with no ischemia (n = 10), while IGF-II and IGFBP-2 were elevated 2.7- and 4.3-fold (P < 0.01). Within the rubeosis group similar changes were observed independently of the cause of ischemia, which was central vein occlusion, ischemic ophthalmopathy, or intraocular tumor in seven cases and diabetic retinopathy in three samples from two patients. Vitreous from patients with proliferative diabetic retinopathy but without rubeosis (n = 16) contained 2.5- and 2.2-fold elevated levels of IGF-I and of IGFBP-2 (P < 0.05), while IGF-II and IGFBP-3 were increased 1.4- and 1.6-fold, which was not significant. We conclude that: (a) ischemia appears to be a strong stimulus for the local production of IGF-I and -II and of IGFBP-2 and -3 in the eye. (b) Changes in IGF-I and IGFBP-2 in proliferative diabetic retinopathy may be secondary to local ischemia rather than being specific for diabetic retinopathy. (c) IGF-I and IGFBP-3 may play a role in mediating angioneogenesis in the eye.

Elevated Plasma Levels of Transforming Growth Factor-β1 in NIDDM

OBJECTIVE Transforming growth factor-β (TGF-β) is a potent inducer of extracellular matrix production and of fibrogenesis and has been associated with the occurrence of diabetic micro- and macrovascular complications. Our aim was to determine whether circulating levels of TGF-β1 are altered in NIDDM and, if so, whether they are correlated with blood glucose and show an association with diabetic complications. RESEARCH DESIGN AND METHODS Plasma levels of TGF-β1 were determined by enzyme-linked immunosorbent assay in 44 NIDDM patients and 28 control subjects of comparable age and weight and were correlated with parameters of metabolic control and the occurrence of micro- and macrovascular complications. RESULTS TGF-β1 was significantly elevated in NIDDM (7.9 ± 1.0 ng/ml), as compared with control subjects (3.1 ± 0.4 ng/ml, P < 0.001) and correlated with glycosylated hemoglobin (r2 = 0.42; P < 0.001). Thrombocyte levels of TGF-β1 were similar in control subjects (54 ± 7 pg/ml, n = 16) and diabetic patients (61.6 ± 18 pg/ml, n = 13; P = 0.357). Elevated TGF-β1 levels were associated with retinopathy and neuropathy. CONCLUSIONS We conclude that plasma levels of TGF-β1 are elevated in NIDDM patients and may be related to average blood glucose. Preliminary data suggest that they may contribute to the occurrence of diabetic complications.

Regulation of protein kinase C by short term hyperglycaemia in human platelets in vivo and in vitro

Aims/hypothesis. Postprandial hyperglycaemia carries an increased risk of macrovascular disease even without Type II (non-insulin-dependent) diabetes mellitus. Chronic hyperglycaemia activates protein kinase C (PKC) in vitro and in vivo but it is not known whether PKC is regulated by short-term postprandial hyperglycaemia in vivo in humans. We investigated whether PKC is regulated in vivo in hyperglycaemic and hyperinsulinaemic infusion tests and correlated the results to stimulations in vitro. Methods. Protein kinase C regulation was measured in platelets obtained from 8 healthy subjects who were infused with glucose and insulin for 2 h attaining peak concentrations of 16 mmol/l glucose and in platelets from 8 healthy young subjects, 8 older subjects without diabetes, and 10 older subjects with Type II diabetes after incubation in vitro with 16 mmol/l glucose or glucose and insulin. For precise quantification, a shortened PKC β1 standard protein was generated by bacterial expression and PKC α, β1, β2 and δ isoenzyme values were measured by immunoblot analyses. Results. Hyperglycaemic and hyperinsulinaemic in vivo tests increased the amounts of PKC α, β1 and β2 in the membrane fraction of platelets to 225 ± 87 %, 164 ± 22 % and 302 ± 135 %, respectively, when compared with the baseline values in young healthy volunteers (n = 8, p < 0.05). The expression of PKC δ did not change. In comparison to the recombinant PKC β1 standard protein, 5 ng PKC β1/μg protein was measured before the test and 2 ng/μg were translocated to the membrane fraction after the infusion. No change in the absolute amount of PKC β1 was detected. In contrast, after incubation in vitro PKC was not regulated by glucose or glucose and insulin in 8 young healthy subjects (age 26 ± 0.7 years) and in 8 older, healthy subjects (age 64,8 ± 4 years) although 100 nmol/l 12-O-tetradecanoylphorbol 13-acetate caused maximal activation. In marked contrast, PKC β1 and PKC β2, but not PKC α or PKC δ, were increased in vitro in the membrane fraction by 292 ± 61 % and 432 ± 88 % (p < 0.05) in 10 subjects with Type II diabetes mellitus matched for age, sex and BMI. Conclusion/interpretation. We found that short-term hyperglycaemia activates PKC α, β1 and β2 in platelets of healthy persons making them potential candidates for mediating the increased cardiovascular risk of postprandial hyperglycaemia. Hyperglycaemia and hyperinsulinaemia did not cause short-term activation of PKC in platelets in vitro suggesting the existence of additional stimuli. Subjects with Type II diabetes showed a markedly altered reactivity of platelet PKC β in vitro indicating some diabetes-related regulation. [Diabetologia (2001) 44: 188–195]

Growth Factor Alterations in Advanced Diabetic Retinopathy: A Possible Role of Blood Retina Barrier Breakdown

Chronic hyperglycemia may cause growth factor alterations that are likely to participate in tissue remodeling typical for diabetic late complications. However, few details of such events are known. The ocular vitreous fluid allows studies of growth factor levels in human eyes (after vitrectomy). The vitreous is highly inert and protected by the blood-retina barrier and thus probably reflects growth factor production by the normal retina. Vitreous from patients with proliferative diabetic retinopathy (PDR) was compared with vitreous obtained from patients with nonproliferative eye disease and with vitreous from patients without diabetes but with marked neovascular proliferations due to ischemia. This design permits us to distinguish diabetes-related from non-diabetes-related alterations. Insulin-like growth factor I (IGF-I), IGF-II, IGF binding protein 2 (IGFBP-2), and IGFBP-3 were elevated 3-to 13-fold in nondiabetic retinal ischemia and 1.5- to 3-fold in PDR, indicating that the changes were not restricted to diabetes. These changes may partially be explained by leakage of serum into the vitreous, since IGFs and IGFBPs are 20- to 50-fold higher in serum than in vitreous, and vitreous protein content was 1.5-fold elevated in PDR subjects and 5-fold in ischemia patients compared with control subjects. TGF-β is a proposed antiangiogenic factor in the eye. TGF-β2 was the predominant subtype in vitreous, and its total amount was not altered in PDR patients. More importantly, the active fraction of TGF-β was decreased by 30 and 70% in PDR and nondiabetic retinal ischemia patients, respectively. Since plasmin may control TGF-β activation, the serum protein α2-antiplasmin was measured and found to be significantly elevated to 150 and 250% of control values in PDR and ischemia patients, respectively. Thus, influx of serum proteins due to microvascular disturbances and hypoxia is proposed as a possible cause for vitreous alterations of IGF-I and of active TGF-β. These changes seem to occur late in the sequence of events leading to PDR and are not specific for diabetes, but they were also observed in other diseases characterized by retinal hypoxia.

Release of the angiogenesis inhibitor angiostatin in patients with proliferative diabetic retinopathy : association with retinal photocoagulation

Aims/hypothesis. Proliferative diabetic retinopathy is a major debilitating disease causing most cases of blindness in humans in the Western world. Photocoagulation is the established therapy of proliferative diabetic retinopathy, although the molecular mechanisms of its effects are still not known. Recently angiostatin has been characterized as a potent inhibitor of neovascularization. Apart from a possible down-regulation of angiogenic cytokines, release of angiostatin could initiate the anti-angiogenic effects of retinal photocoagulation.¶Methods. We investigated the regulation of angiostatin and the angiogenic cytokines vascular endothelial growth factor and basic fibroblast growth factor in vivo by comparing vitreal concentrations of 18 control patients and 34 patients with proliferative diabetic retinopathy with and without previous photocoagulation. Concentrations of basic fibroblast growth factor and angiostatin were additionally measured in serum, while vascular endothelial growth factor is known to be regulated locally in the eye. Cytokines were measured by immunological methods.¶Results. Angiostatin could be detected in 2 out of 18 control patients and in 25 out of 34 diabetic patients (p < 0.00 001). Most importantly, production of angiostatin in human vitreous correlated significantly with previous retinal photocoagulation (p < 0.0001) in patients with proliferative diabetic retinopathy. Only two patients (one control and one diabetic) had detectable serum concentrations of angiostatin. Additionally patients with proliferative diabetic retinopathy and with previous photocoagulation had significantly lower concentrations of vascular endothelial growth factor (0.9 ± 0.1 ng/ml; p < 0.0001) than diabetic patients without previous photocoagulation (4.0 ± 0.8 ng/ml). The investigation of vitreal and serum basic fibroblast growth factor concentrations yielded no significant differences between the groups.¶Conclusion/interpretation. Angiostatin is not a regularly expressed angiogenesis inhibitor in human vitreous. The alterations we observed suggest that local release of angiostatin and down-regulation of vascular endothelial growth factor mediate the therapeutic effects of retinal photocoagulation in proliferative diabetic retinopathy. [Diabetologia (2000) 43: 1404–1407]

Determinants and estimation of healing times in diabetic foot ulcers

AIMS To assess the wound size reduction and time course for healing and to establish equations to predict the time course of wound healing in neuropathic, neuroischemic, and ischemic diabetic foot ulcers. METHODS This prospective study evaluates wound healing over at least a 10-week period in 31 Type 1 or Type 2 diabetic patients with plantar foot ulcers. Thirteen consecutive diabetic patients with neuropathic foot ulceration, 10 consecutive diabetic patients with neuroischemic ulceration, and 8 diabetic patients with peripheral occlusive vascular disease were selected for the study. All patients received identical ulcer wound care including use of proper footwear, non-weight-bearing limb support, use of appropriate antibiotics, debridement, tight control of serum glucose levels, and careful monitoring of the ulcer. Ulcer healing was assessed by planimetric measurement of the wound area every second week until wound healing. The time course of wound healing was calculated by the daily wound radius reduction. RESULTS The wound area (mean+/-S.E.) in the patients with neuropathic foot ulceration was 61.2+/-17.1 at the beginning and 3.2+/-1.5 mm(2) after 70 days (P=.005). The wound radius decreased by 0.045 mm (95% confidence interval [CI] 0.039-0.055) per day, with most of the wound healing being achieved between the first and seventh week of ulcer care. The average healing time was 77.7 (95% CI 62-93) days. In the neuroischemic group, the initial average wound area was 26.6+/-7.0 mm(2), and 6.25+/-1.7 mm(2) after 10 weeks (P=.007). The wound radius reduction was 0.019 mm/day (95% CI 0.017-0.023) with an average healing time of 123.4 (95% CI 101-145) days. The diabetic patients with peripheral occlusive vascular disease had an average wound size of 32.6+/-13.1 at the beginning and 23.9+/-10.7 mm(2) after 70 days of ulcer care (P=.06). The daily wound radius reduction was 0.0065 mm (95% CI 0.0039-0.0091). Average ulcer duration was 133 (95% CI 116-149) days, but three of eight patients achieved no wound healing. CONCLUSIONS Providing standard care, the time course of wound healing in diabetic foot ulcers is predominantly determined by etiologic factors, and less by wound size. Taking wound etiology and wound radius into account, the expected healing time can reliably be estimated in neuropathic and neuroischemic ulcers.

Cytomegalovirus infection in systemic necrotizing vasculitis: causative agent or opportunistic infection?

Abstract We report on a 69-year-old woman who presented with myalgia, hearing impairment, fever, night sweats, weight loss, muscular weakness, paresthesia, hypesthesia, and hypalgesia. Sural nerve biopsy showed demyelinative and axonal polyneuropathy due to necrotizing vasculitis with fibrinoid necrosis. A positive test for antineutrophil cytoplasmic antibodies (ANCA) with a perinuclear immunofluorescence pattern directed against myeloperoxidase was more suggestive of microscopic polyangiitis (MPA) than of polyarteritis nodosa (PAN), the possible differential diagnoses. In addition, positive tests for cytomegalovirus (CMV) antibodies (immunoglobulin (Ig)M and IgG) and the detection of CMV-DNA in sputum specimens by polymerase chain reaction (PCR) were indicative of active CMV infection. Treatment with ganciclovir and anti-CMV immunoglobulin in addition to prednisolone medication for 6 months resulted in rapid improvement of the clinical symptoms without relapse. CMV infection has been described to be related to ANCA-associated vasculitis in non-immunocompromized patients and may be either a causative agent or an opportunistic infection. Identification of a viral etiology in patients with atypical ANCA-associated vasculitides may lead to different, less aggressive treatment approaches, including antiviral therapy.

Impaired 0.1-Hz vasomotion assessed by laser Doppler anemometry as an early index of peripheral sympathetic neuropathy in diabetes

Impairment of 0.1-Hz vasomotion, which was found in diabetic patients, was suggested to be an early index of sympathetic dysfunction. We studied the relationships between alterations in vasomotion and both cardiac autonomic and sensory neuropathy. Twenty type 1 and 22 type 2 diabetic patients were investigated. Vasomotion was recorded in single capillaries at the dorsal middle phalangeal area of the left ring finger by means of laser Doppler anemometry. Cardiac autonomic neuropathy was assessed by spectral analysis of heart rate variation during rest and recording heart rate responses to deep breathing and Valsalva manoeuvre. Sensory neuropathy was investigated by measuring heat pain, vibration, and thermal sensory thresholds. Impaired vasomotion was more often (82.4%) found in diabetic patients with at least one altered cardiac autonomic test, but also in 47.1% of those with all of these tests being normal (P = 0.035). Both heart rate variation coefficient during rest (r = 0.40, P = 0.045) and Valsalva ratio (r = 0.41, P = 0.037) correlated positively with amplitudes of vasomotion in type 1 diabetic patients. The prevalence of impaired vasomotion was not higher in patients with sensory neuropathy compared to those with normal sensory functions. A disturbed warm sensation was only found in 2 patients and none had an abnormal heat pain threshold. Our data indicate that impairment of 0.1-Hz vasomotion precedes parasympathetic neuropathy, assessed by heart rate variation tests, and abnormalities in warm and heat pain sensation. Reduction of arteriolar vasomotion, detected by laser Doppler anemometry, might be an early index of sympathetic dysfunction, because it correlates with disturbances in those cardiac autonomic tests, which are at least in part under sympathetic control.

Anti-MPO-ANCA-Positive Microscopic Polyangiitis following Subacute Bacterial Endocarditis

Abstract: Although infectious agents such as Staphylococcus aureus have been implicated in the pathogenesis of Wegener’s granulomatosis, the role of bacterial infections in the pathogenesis of other types of small-vessel vasculitides associated with antineutrophil cytoplasmic antibodies (ANCA) is less clear. We describe a patient who developed a non-granulomatous necrotising small vessel vasculitis and perinuclear ANCA (p-ANCA) directed against myeloperoxidase (MPO) after recurrent episodes of bacterial endocarditis due to Staph. aureus. Although cytoplasmic ANCA (c-ANCA) directed against proteinase 3 have been reported in single patients with bacterial endocarditis, to our knowledge this patient is the first reported case of an anti-MPO-ANCA positive systemic vasculitis following bacterial endocarditis.

Activation of transforming growth factor-β1 in diabetic kidney disease

Recent data have suggested that certain growth factors and cytokines are involved in the development of diabetic nephropathy. The aim of this study was to investigate whether circulating transforming growth factor beta 1 (TGF-beta1) and tumor necrosis factor alpha (TNF-alpha) are associated with diabetic kidney disease. Serum levels of active and total TGF-beta1 and TNF-alpha were measured in type 2 diabetic patients with nephropathy (n = 23) or without (n = 35) and normoglycemic controls (n = 12). Serum levels of circulating active TGF-beta1 were significantly higher in patients with diabetic nephropathy (0.43 +/- 0.06 ng x mL(-1)) compared with diabetic patients without renal involvement (0.23 +/- 0.03 ng x mL(-1), P = .002) and healthy controls (0.24 +/- 0.03 ng x mL(-1), P= .001), whereas the levels of total (active + latent) TGF-beta1 were not different between the subgroups. Active TGF-beta1 concentrations were correlated with urinary albumin excretion (r = .49, P < .003) and serum creatinine (r= .55, P < .01). Sera from patients with type 2 diabetes contained significantly more TNF-alpha than sera from normoglycemic controls (3.07 +/- 0.24 v 1.65 +/- 0.20 pg x mL(-1), P = .001). However, the comparison of serum TNF-alpha concentrations between microalbuminuric and normoalbuminuric diabetic patients showed no significant difference (3.21 +/- 0.28 v 2.97 +/- 0.34 pg x mL(-1), P = .12). In conclusion, type 2 diabetic patients with diabetic nephropathy exhibit increased activation of TGF-beta1, in serum, suggesting an association between circulating TGF-beta1 activity and the development of renal disease.