In a cross-sectional study we identified increased serum kallistatin levels in Type 1 diabetic patients and in Type 1 diabetic patients with microvascular complications vs. age and gender-matched non-diabetic subjects. Further, kallistatin levels were elevated in diabetic subjects with hypertension vs. those without. There were no statistically significant differences in kallistatin levels between complication-free Type 1 diabetic patients and healthy subjects or between diabetic subjects with or without vascular complications. In the diabetic subjects considered as a single group, kallistatin levels were associated with renal dysfunction, total and LDL-cholesterol levels, and inversely with SAE, reflecting vascular dysfunction. Kallistatin levels in diabetes did not correlate with other lipids, glycemia, BMI, smoking, or measures of inflammation or oxidative stress. Common statistically independent determinants of kallistatin levels in all subjects and in the separate diabetes and control groups were renal function (most strongly) and cholesterol, with age, hepatic and vascular function also being related to kallistatin level variability in the combined group.
Functions, sources, and levels of kallistatin
Kallistatin has vasodilatory, anti-angiogenic, anti-inflammatory and anti-oxidant effects [7–10, 12, 13]. It is localized in many human tissues, including eye, kidney, liver, heart, arteries and veins, atheroma, blood cells and body fluids [7, 14, 15, 17]. The relative contributions of various cell and tissue types to circulating kallistatin levels in health and disease is unknown, but several studies support liver as a major source [7, 14, 25]. Hepatocytes secrete kallistatin [7, 14, 25] and in a small cross-sectional study of cirrhosis patients, circulating kallistatin levels were ≈30% that of healthy people . In our study there were correlations between kallistatin levels and normal range liver function tests, perhaps in keeping with a predominant hepatic origin of serum kallistatin.
Increased kallistatin in diabetes complications
To the best of our knowledge, the only prior study of kallistatin in human diabetes is by coauthor J-X Ma et al in which immunoreactive kallistatin levels in vitreous fluid from 18 patients with diabetic retinopathy were significantly lower compared to 17 non-diabetic subjects . We now demonstrate higher serum kallistatin levels in Type 1 diabetic patients with vascular complications, which include proliferative retinopathy. We have noted a similar pattern with another serpin, Pigment Epithelium Derived Factor (PEDF), with low vitreous fluid levels in diabetic retinopathy patients and high serum levels with microvascular complications . In the present study, kallistatin levels also related negatively to renal function, which could be due to reduced renal excretion or increased production, or both. Kallistatin has been localized in human urine [14, 26] and in kidneys [7, 14, 27], where it is thought to regulate salt and water handling, renal perfusion and blood pressure, and to reduce intra-renal fibrosis, inflammation, and oxidative stress [7, 12, 14, 28]. Apart from the current study, plasma, serum, renal tissue or urine kallistatin levels have not been reported in human diabetes or in other renal diseases. It may be that kallistatin levels rise in response to renal disease and proteinuria together with other circulating proteins of hepatic origin . Urinary kallistatin excretion, not measured in this study as there were no urine kallistatin ELISA assays, merits future study.
Kallistatin levels are associated with impaired vascular health
We observed positive associations between kallistatin and systolic blood pressure and pulse pressure, and an inverse correlation with SAE. Furthermore, serum kallistatin levels were higher in diabetic patients with vs. without diagnosed hypertension, even after statistical correction for renal dysfunction, and were also associated with impaired renal function by several measures. One possibility is that elevated kallistatin levels may be compensatory to mitigate the high blood pressure and endothelial dysfunction, as kallistatin is a potent vasodilator [7, 10] and lowers blood pressure [30, 31]. Other animal and isolated vascular cell experiments support a role of kallistatin in vascular biology, including vascular and cardiac remodeling [7, 11] and angiogenesis [8, 13]. Kallistatin increases in vitro growth, proliferation and migration of vascular smooth muscle cells and inhibits in vitro proliferation, migration, and adhesion of vascular endothelial cells . In rats, balloon angioplasty markedly increased kallistatin mRNA and protein expression in injured vessels, which along with neointima formation was attenuated by local delivery of kallistatin antisense cDNA . Kallistatin also inhibits angiogenesis in in vivo rat models of hind-limb ischemia and tumor growth .
Kallistatin and inflammation and oxidative stress
The higher serum levels of inflammation markers (WCC, ESR and cell adhesion molecules) in diabetes and/or its complications in the present study are in keeping with other publications [20, 32–34]. In our study, except for a positive correlation with WCC in controls on adjusted analyses, kallistatin levels were not significantly related to inflammation or oxidative stress measures. This contrasts with other literature [9, 12, 35, 36]. In people with (inflammatory) rheumatoid arthritis, plasma and joint kallistatin levels were increased relative to osteoarthritis patients . In animal studies, kallistatin gene delivery has anti-inflammatory and anti-oxidant effects, inhibiting renal inflammation including renal CAM expression in a rat renal disease model , inhibiting inflammation and apoptosis in acute myocardial ischemia-reperfusion injury , and reducing inflammation and joint injury in rat arthritis models . Kallistatin levels decline during sepsis and severe inflammation, as markedly lower circulating kallistatin levels have been reported in humans with sepsis  and in necrotic acute pancreatitis . In animal models hepatic kallistatin expression is reduced by lipopolysaccharide (LPS) , and transgenic mice overexpressing human kallistatin have lower LPS-induced mortality . These previous studies support that kallistatin is an anti-inflammatory factor, but our present cross-sectional clinical study, in which serum kallistatin levels are not strongly associated with serum inflammation markers do not support a major anti-inflammatory role. This may relate to the level of inflammation in diabetes being relatively low, local tissue anti-inflammatory effects not being well-reflected by circulating measures, or to opposing effects of the effects of inflammation (decreasing kallistatin) and of renal and vascular dysfunction (increasing kallistatin).
Angiogenesis and arteriogenesis in atherosclerosis
Neovascularisation, including angiogenesis and arteriogenesis (the rapid proliferation of pre-existing arterial vessels, which have a mature tunica media), is required to heal wounds and for collateral circulation development in ischemic tissues , common problems in diabetic patients [1, 40]. However, angiogenesis within atheromatous plaques may be deleterious as leaky new vessel formation may promote inflammation, plaque growth, hemorrhage, instability, and rupture [40–43]. There is likely a delicate balance between pro- and anti-angiogenic factors, which likely varies at the different stages of blood vessel formation and repair and plaque formation, stability and regression. It is currently controversial as to whether pro- or anti-angiogenic factor based therapies will benefit atherosclerosis [44–46]. PEDF, another member of the serpin family, is now undergoing evaluation as a potential therapeutic agent for ocular angiogenesis . The specific role and potential therapeutic effects of kallistatin in vascular disease, including atheroma progression and plaque stability, and specifically in the context of diabetes, remains unclear.
Study limitations and future research
The limitations of a cross-sectional study are recognized, and kallistatin responses may vary by tissue in ways not necessarily reflected in serum levels. Longitudinal studies of kallistatin and the various types of diabetic complications are desirable. As higher kallistatin levels were inversely related to renal function, even in our study groups with relatively normal renal function, such as reflected by serum creatinine and urea, kallistatin levels in urine and in serum of people with different types and degrees of renal damage are merited. As we observed associations between kallistatin and blood pressure, vascular dysfunction, lipids and renal function, studies pre- and post-interventions targeting these factors are merited. Wound healing studies, mechanistic vascular reactivity studies involving diabetic animals, isolated vessels and plaque and cell culture models are relevant. Future studies may utilize additional measures of inflammation and oxidative stress.