Ulrich M. Vischer

von Willebrand factor, endothelial dysfunction, and cardiovascular disease

Summary.  von Willebrand factor (VWF), a glycoprotein involved in arterial thrombus formation, is released into the circulation by secretion from endothelial cells. Plasma VWF levels are determined by genetic factors including ABO blood groups and VWF mutations, and by non‐genetic factors including aging, impaired nitric oxide production, inflammation, free radical production and diabetes. Plasma VWF levels have been proposed as a risk factor for cardiovascular events. Although they are only weakly associated with the risk of coronary heart disease (CHD) in the general population, they are a more promising CHD risk factor in high‐risk populations with previous cardiovascular events, diabetes or old age. However, is it still unclear whether VWF levels directly determine the rate and severity of arterial thrombus formation or whether they merely reflect alteration in other endothelial functions. The future status of VWF levels as a cardiovascular risk factor depends on additional studies on the genetic determinants of both VWF levels and cardiovascular outcomes. Further studies on VWF levels as a predictor of the risk of stroke (rather than CHD) in elderly or other high‐risk population are also promising. Such studies could lead to the clinical use of plasma VWF levels to refine the estimation of the cardiovascular risk and of the expected benefit of antithrombotic agents.

Vasopressin-induced von Willebrand factor secretion from endothelial cells involves V2 receptors and cAMP.

Vasopressin and its analogue 1-deamino-8-D-arginine vasopressin (DDAVP) are known to raise plasma von Willebrand factor (vWF) levels. DDAVP is used as a hemostatic agent for the treatment of von Willebrands disease. However, its cellular mechanisms of action have not been elucidated. DDAVP, a specific agonist for the vasopressin V2 receptor (V2R), exerts its antidiuretic effect via a rise in cAMP in kidney collecting ducts. We tested the hypothesis that DDAVP induces vWF secretion by binding to V2R and activating cAMP-mediated signaling in endothelial cells. vWF secretion from human umbilical vein endothelial cells (HUVECs) can be mediated by cAMP, but DDAVP is ineffective, presumably due to the absence of V2R. We report that DDAVP stimulates vWF secretion in a cAMP-dependent manner in HUVECs after transfection of the V2R. In addition, vasopressin and DDAVP induce vWF secretion in human lung microvascular endothelial cells (HMVEC-L). These cells (but not HUVECs) express endogenous V2R, as shown by RT-PCR. Vasopressin-induced vWF secretion is mimicked by DDAVP and inhibited by the selective V2R antagonist SR121463B. It is mediated by cAMP, since it is inhibited by the protein kinase A inhibitor Rp-8CPT-cAMPS. These results indicate that vasopressin induces cAMP-mediated vWF secretion by a direct effect on endothelial cells. They also demonstrate functional expression of V2R in endothelial cells, and provide a cellular mechanism for the hemostatic effects of DDAVP.

Long-term effects of combined hormone replacement therapy on markers of endothelial function and inflammatory activity in healthy postmenopausal women

OBJECTIVE To study the effects of combined hormone replacement therapy on markers of endothelial function and inflammatory activity. DESIGN Prospective, randomized, controlled study. SETTING Academic hospital. PATIENT(S) Healthy postmenopausal women with an intact uterus. INTERVENTION(S) For the first 12 months, the hormone replacement therapy group (n = 14) received oral E2, 1 mg daily, sequentially combined with 5 or 10 mg of dydrogesterone. Thereafter, they received oral E2, 2 mg daily, sequentially combined with 10 mg of dydrogesterone. The control group (n = 13) received no treatment. Data were collected at baseline and at 3, 12, and 15 months. MAIN OUTCOME MEASURE(S) Parameters of endothelial function and inflammatory activity. RESULT(S) During 12 months of follow-up, we observed decreases of 15% in plasma levels of endothelin-l, of 21% in soluble thrombomodulin, of 14% in von Willebrand factor, and of 12% in clottable fibrinogen in the hormone replacement therapy group compared with the control group. There was a 5% decrease in soluble E-selectin tevels. All significant changes were observed by 3 months and sustained after 15 months. Brachial artery flow-mediated vasodilatation and C-reactive protein levels did not change significantly. CONCLUSION(S) Long-term combined hormone replacement therapy with E2 and dydrogesterone in healthy women was associated with sustained improvement in some aspects of endothelial function and in clottable fibrinogen levels.

Regulated von Willebrand Factor Secretion Is Associated With Agonist-Specific Patterns of Cytoskeletal Remodeling in Cultured Endothelial Cells

von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis, is stored and released from endothelial secretory granules called Weibel-Palade bodies. Regulated secretion occurs in reaction either to [Ca(2+)](i)-raising agents (histamine or thrombin) or to cAMP-raising agents (epinephrine, adenosine, or forskolin). We investigated the pattern of release and the cytoskeletal requirements for secretion in response to these 2 classes of agonists. Secretion induced by [Ca(2+)](i)-raising agents involves peripheral and central granules and is inhibited by colchicine-induced microtubule disruption. It is accompanied by Rho-dependent stress fiber formation and cell retraction. Secretion and remodeling occur in the same individual cells. However, secretion is potentiated by cytochalasin E and C3 toxin, indicating that stress fiber formation antagonizes vWF secretion. In contrast, vWF secretion induced by cAMP-raising agents involves the release of only peripheral granules (implying less vWF release on a per cell basis) and is not inhibited by microtubule disruption. cAMP-mediated secretion is accompanied by disruption of stress fibers, strengthening of the cortical actin rim, and preservation of cell-cell contacts. It is unaffected by cytochalasins or C3 toxin. In contrast to [Ca(2+)](i)-raising agents, cAMP-raising agents induce secretion without cell retraction/intercellular gap formation. Thus, they are likely to play a physiological role in the regulation of endothelial vWF secretion and, therefore, of plasma vWF levels.

Storage of Tissue-Type Plasminogen Activator in Weibel-Palade Bodies of Human Endothelial Cells

Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone-containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.

Diabetic foot disease in the elderly.

Elderly diabetic patients are particularly burdened by foot disease. The main causes for foot disease are peripheral neuropathy, foot deformities and peripheral arterial disease (PAD). Other risk factors include poor vision, gait abnormalities, reduced mobility an medical co-morbidities. The risk of major amputations increases with age, along with the increased prevalence of these risk factors. Th true risk of amputation and other burdens of foot disease in the elderly are likely underestimated by current epidemiological data. Th prevalence of neuropathy, foot deformities and PAD as well as the risk of amputation all increase with age even in non-diabetic patients. The principles of prevention and management of diabetic foot disease may also apply to large segments of the elderly non-diabetic population. Foot ulcer prevention relies on the identification of high risk patients and avoidance of triggering events, such as ill-fitting shoes, walking barefoot or poor self-care. PAD is a major cause of amputation and should be prevented by lifelong attention to glycaemic control, treatment of hypertension and dyslipidemia, and avoidance of smoking. The treatment of foot ulcers relies on pressure relief (off-loading), wound debridement, and treatment of infection and ischemia. It requires an individualized approach considering the patients co-morbidities and functional status. Off-loading remains essential, but devices such as total contact casts or crutches can only rarely be implemented. However, providing adapted standard foot-wear and insisting on its consistent use even at home is often effective. The benefits of aggressive vascular or orthopaedic surgery should be weighed against the risks of prolonged hospitalisation and resulting functional decline. Greater attention to prevention and individualized care are needed to reduce the burden of diabetic foot disease in the elderly.

The high prevalence of malnutrition in elderly diabetic patients: implications for anti-diabetic drug treatments.

Diabet. Med. 27, 918–924 (2010)

Key priorities in managing glucose control in older people with diabetes

Older people with diabetes represent a major and increasing proportion of our elderly population and their care requires better organisation. Targets for risk factor control and pathways of care must be adjusted to the subjects general health status. It is thus advisable to screen for frailty. We have carried out a detailed literature review of the studies published on diabetes in older people since 1990. Studies were considered if they included groups or subgroups of diabetic patients > 65 years old. This review discusses the elaboration of general targets for care, the approach to risk factor control, the screening and the specific prevention or management of complications, the integration of geriatric concepts in diabetes care and the specificity of education with respect to frailty status.

Protein kinase C‐δ mediates von Willebrand factor secretion from endothelial cells in response to vascular endothelial growth factor (VEGF) but not histamine

Summary.  Background: Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of exocytosis in many secretory cell types. Objectives: We investigated the role of PKC and the interactions between PKC and Ca2+ signaling in both VEGF‐induced and histamine‐induced VWF secretion from human umbilical vein endothelial cells (HUVECs). Results: Several PKC inhibitors (staurosporine, Ro31‐8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF‐induced but not histamine‐induced VWF secretion. PKC‐α and novel PKCs (PKC‐δ, PKC‐ε, and PKC‐η), but not PKC‐β, are expressed in HUVECs. Both VEGF and histamine activate PKC‐δ. However, gene inactivation experiments using small interfering RNA indicate that PKC‐δ (but not PKC‐α) is involved in the regulation of VEGF‐induced but not histamine‐induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca2+ ([Ca2+]c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF‐induced secretion is largely preserved when the rise in [Ca2+]c is prevented by BAPTA‐AM. Conclusions: Our study identifies striking agonist specificities in signal–secretion coupling. Histamine‐induced secretion is dependent on [Ca2+]c but not PKC, whereas VEGF‐induced secretion is largely dependent on PKC‐δ and significantly less on [Ca2+]c. Our data firmly establish the key role of PKC‐δ in VEGF‐induced VWF release, but suggest that a third, VEGF‐specific, signaling intermediate is required as a PKC‐δ coactivator.

Dopamine modulates von Willebrand factor secretion in endothelial cells via D2–D4 receptors

Summary.  Objective: von Willebrand factor (VWF) is acutely released from endothelial cells in response to numerous calcium‐raising agents (e.g. thrombin, histamine) and cAMP‐raising agents (e.g. epinephrine, adenosine, vasopressin). In contrast, very few inhibitors of endothelial VWF secretion have been described. The neurotransmitter dopamine is a modulator of exocytosis in several endocrine cells, and is possibly involved in the regulation of several endothelial cell functions. We therefore investigated the effect of dopamine on endothelial VWF secretion. Results: Dopamine, D2/D3‐ and D4‐specific agonists inhibited histamine‐ but not thrombin‐induced VWF secretion. Expression of dopamine D2, D3 and D4 receptors was demonstrated by reverse transcription polymerase chain reaction (RT‐PCR) in both human aortic (HAEC) and umbilical vein (HUVEC) endothelial cells. D2–D4 agonists did not inhibit histamine‐induced rise in [Ca2+]i: they inhibited histamine‐induced secretion even in the absence of extracellular calcium. Thus, the dopamine effects are not mediated by [Ca2+]i‐dependent signalling. D2/D3‐ and D4‐specific agonists inhibited neither the rise in cAMP nor VWF secretion in response to epinephrine and adenosine, arguing against an effect on cAMP‐mediated signalling. D1 and D5 receptors were not detected in HAEC or HUVEC by RT‐PCR, and the D1/D5‐specific agonist SKF 38 393 failed to modulate VWF secretion, arguing against a role for these receptors in endothelial exocytosis. Conclusions: Dopamine inhibits histamine‐induced endothelial exocytosis by activating D2–D4 receptor, via a mechanism distinct from [Ca2+]i‐or cAMP‐mediated signaling. In contrast, D1 and D5 receptors are not functionally expressed in cultured endothelial cells. Dopamine agonists may be useful as inhibitors of endothelial activation in inflammation and cardiovascular disease.