Full Text HL-97-014 ENDOTHELIAL DYSFUNCTION IN HIV INFECTION NIH Guide, Volume 26, Number 27, August 15, 1997 RFA: HL-97-014 P.T. Keywords: National Heart, Lung and Blood Institute Letter of Intent Receipt Date: January 5, 1998 Application Receipt Date: March 26, 1998 PURPOSE This solicitation invites research grants focused on how HIV infection alters the expression of endothelial cell genes thereby modifying the normal structure and function of the endothelium and exposing organs to HIV-infected cells and to circulatory factors that could cause damage. Ultimately, the goal of this solicitation is to contribute to knowledge that might lead to new approaches to prevent HIV associated dysfunction and degeneration caused by HIV to vital organs, including lungs, heart, bone marrow, and the vasculature. HEALTHY PEOPLE 2000 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2000," a PHS-led national activity for setting priority areas. This Request for Applications (RFA), "Endothelial Dysfunction in HIV Infection" is related to the priority area of HIV Infection. Potential applicants may obtain a copy of "Healthy People 2000" (Full Report: Stock No. 017-001-00474-0) or "Healthy People 2000" (Summary Report: Stock No. 017-001-00473-1) through the Superintendent of Documents, Government Printing Office, Washington, DC 20402-9325 (telephone 202-512-1800). ELIGIBILITY REQUIREMENTS Applications may be submitted by domestic, for-profit and non-profit organizations, public and private, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal government. Racial/ethnic minority individuals, women, and persons with disabilities are encouraged to apply as Principal Investigators. Applications from foreign institutions will not be accepted. However, subcontracts to foreign institutions are allowable, with sufficient justification. All current policies and requirements that govern the research grant programs of the NIH will apply to grants awarded under the RFA. Awards under this announcement to foreign institutions will be made only for research of very unusual merit, need, and promise, and in accordance with Public Health Service policy governing such awards. Among the disciplines and expertise that may be appropriate for this research program are cell biology, molecular biology, developmental biology, biochemistry, genetics, immunology, pathology, virology, physiology, cardiology, hematology, and pulmonology. MECHANISM OF SUPPORT This RFA will use the NIH individual research project grant (R01) mechanism of support. Investigators without prior R29 or R01 support are encouraged to apply for this RFA and to identify their status in a cover letter. However, specific application instructions have been modified to reflect "MODULAR GRANT" and "JUST-IN-TIME" stream-lining efforts being examined by the NIH. The modular grant concept establishes specific modules in which direct costs may be requested as well as a maximum level for requested budgets. Only limited budgetary information is required under this approach. The just-in- time concept allows applicants to submit certain information only when there is a possibility for an award. It is anticipated that these changes will reduce the administrative burden for the applicants, applicant institutions, reviewers and Institute staff. For this RFA, funds must be requested in $25,000 direct cost modules and a maximum of eight modules ($200,000 direct costs) per year may be requested. A feature of the modular grant concept is that no escalation is provided for future years, and all anticipated expenses for all years of the project must be included within the number of modules being requested. Only limited budget information will be required and any budget adjustments made by the Initial Review Group will be in modules of $25,000. Instructions for completing the Biographical Sketch have also been modified. In addition, Other Support information and the application Checklist page are not required as part of the initial application. If there is a possibility for an award, necessary Budget, Other Support and Checklist information will be requested by NHLBI staff following the initial review. The APPLICATION PROCEDURES section of the RFA provides specific details of modifications to the standard PHS 398 application kit instructions. Responsibility for the planning, direction, and execution of the proposed project will be solely that of the applicant. This RFA is one-time solicitation. Future unsolicited competing continuation applications will compete with all investigator- initiated applications and be reviewed according to the customary peer review procedures. It is anticipated that support for this program will begin in September, 1998. Administrative adjustments in project period and/or amount may be required at the time of the award. FUNDS AVAILABLE It is anticipated that for fiscal year 1998, approximately $2.0 million total costs will be available for the first year of support for this initiative. Award of grants pursuant to this RFA is contingent upon receipt of such funds for this purpose. It is anticipated that up to eight new grants will be awarded under this program. Applicants may request up to five years of support. The specific number to be funded will, however, depend on the merit and scope of the applications received and on the availability of funds. Direct costs will be awarded in modules of $25,000 less any overlap or other necessary administrative adjustments. Indirect costs will be awarded based on the negotiated rates. RESEARCH OBJECTIVES Background Current treatment of HIV-infected patients has greatly improved prospects for survival due to introduction of drugs to inhibit replication of the virus. A question that remains to be answered is whether reduction in viral burden will result in retardation of, and possibly permit healing of, organ damage. Such damage appears to be, in part, the result of transmission of HIV-infected cells across the endothelium, usually without actual infection of endothelial cells themselves, although whether endothelial cells can be infected by HIV is still open to debate. Under normal circumstances, the endothelial cell barrier plays a critical role in the protection of tissues, but it is clear that in HIV-infected patients this barrier fails, permitting exposure to infections and other sources of damage. Understanding how infected cells and soluble factors present in circulating blood can change gene expression in endothelial cells could lead to new therapeutic options for the treatment of HIV- induced organ damage. The endothelium, which forms a single cell layer throughout the vascular system, performs multiple functions that are often modified by regional location. In addition to providing a balance between vasoconstriction and vasodilation and anti- and pro-coagulation, it is the source of growth factors and cytokines. It expresses adhesion molecules and receptors for local and circulating factors. It also serves to control the influx of macromolecules into the extravascular space. A multitude of circulating factors can activate the endothelium and lead to altered gene expression, thus producing changes in structure and function. En face studies of human aortic endothelium at autopsy have revealed distinct differences between controls and HIV-infected patients. Morphologic changes included disturbances of the regular pattern of the endothelial layer and the presence of smaller cells with rounded nuclei and increased chromatin as well as bizarre-shaped multinucleated cells. Increased adherence of leukocytes, mostly of the monocyte/macrophage lineage, were observed in the endothelium. Endothelial cells from the HIV-infected patients were found to express increased amounts of some adhesion molecules and growth factors and major histocompatibility complex class II antigen (VCAM-1, ELAM-1, HLA-DR antigen, and IL-1beta). On the other hand, in vitro studies have shown that interleukin 2 (IL-2) secretion, which is important for stimulation of T-cell responses, is diminished in HIV-exposed endothelium. How HIV infection causes these changes in endothelial cells and alters their normal immunologic function requires elucidation. HIV-infected monocytes have been shown to exhibit increased adherence to endothelial cells and to traverse the endothelial barrier. One study showed that monocytes accomplished this by producing extracellular matrix-degrading metalloproteinases. Interestingly, tissue inhibitors of metalloproteinases, also produced by monocytes, were increased after HIV infection but not in sufficient quantities to inhibit degradation, suggesting that the balance of these two classes of enzymes is perturbed by HIV. Another study reported that IL-1Beta-activated endothelium is disrupted by elastase secreted by polymorphonuclear cells. Yet another study, focussed on the migration of lymphocytes through monocyte-conditioned endothelium, showed no evidence of disruption of the endothelium. In culture, HIV-infected monocytes were shown not only to exhibit increased adherence to microvascular endothelial cells (MVEC) from skin, brain and lung but also to cause marked morphologic changes in MVEC. These changes included retraction from sites of contact at cell-cell junctions without loss of cell viability as judged by trypan blue exclusion. HIV infection of monocytes increases their expression of the cell adhesion molecules, LFA-1, LFA-2, and LFA-3. Interaction with endothelial cells involves receptors for these molecules as well as adhesion molecules expressed on endothelial cells and their receptors on monocytes. TNFAlpha-activated MVEC showed increased capacity for binding to HIV-infected monocytes and this may be of importance in vivo where high serum levels of TNFAlpha and IL-1 have been documented in HIV-infected patients. TNFAlpha has also been shown to be one of the cytokines produced by monocytes in response to stimulation by HIV-1 glycoprotein 120. The complexity of endothelial cell-HIV relationships is further illustrated by the finding that monocyte contact with endothelial cells upregulates HIV biosynthesis in infected monocytes and may have significant impact on the pathogenesis and tissue distribution of HIV infection. Another source of endothelial damage is the tat protein, which is known to be secreted extracellularly by infected circulating cells. Human umbilical vein endothelial cells in culture have been shown to respond to tat protein by upregulation of IL-6 production and the expression of E-selectin adhesion molecules. E-selectin is specific for endothelial cells and is important for mediating binding of polymorphonuclear cells, monocytes, natural killer cells and CD4+ memory T-cells to the endothelium. IL-6 functions as a potent factor for B-cell maturation and T-cell activation. It also acts on endothelial cells to increase their permeability. The mechanism by which tat protein has these effects on IL-6 and E-selectin production is unclear, although it has been shown to enter cells, to locate to the nucleus and transactivate the HIV long term repeat (LTR). Three mechanisms for tat activation of endothelial cells have been postulated: 1) direct action to enhance transcriptional gene expression of cell factors; 2) indirect enhancement of gene expression by interactions with cell proteins; and 3) initiation of signal transduction by binding to cell membrane receptors. Brain microvascular endothelial cells have also been shown to permit the migration of HIV-1-infected monocytes into the brain by a mechanism that involves induction of adhesion molecules as a result of soluble factors present in the blood. In a recently published paper it was reported that the tat protein contains sequences (amino acids 42-64) similar to sequences of molecules with angiogenic properties, e.g., FGF, VEGF-A, hepatocyte growth factor, and heparin-binding growth factor. A peptide containing this domain was able to induce in vivo angiogenesis that could not be inhibited by metalloproteinase-2. Further studies showed that tat acts through activation of Flk-1/KDR receptor. Flk/KDR is important in vascular development for endothelial cell proliferation and migration but not differentiation. Thus it seems that tat has activated an embryonic gene not normally expressed in mature cells. This activity of the tat protein could account for the aggressiveness of AIDS-associated Kaposi's sarcoma and for the increased incidence of tumors in AIDS patients. It is possible that the altered expression of other genes involved in vascular development and maintenance may hold the key to the morphologic and functional changes that contribute to HIV pathogenesis. For example, recent research has shown that the receptor tyrosine kinase TIE is important in late angiogenesis during development and plays a key role in the survival or proliferation of some, but not all, mature endothelial cell populations. Yet very little research has been conducted along these lines. Another neglected area is that of the lymphatic endothelium and its interactions with lymphocytes. The possibility that other HIV viral proteins might have sequences that are similar to physiologically active peptides or proteins has not been fully explored. In 1992, Parmentier et al. showed that epitopes of HIV regulatory proteins, tat, nef, and rev were expressed in normal human tissues from various sites. They found, for example, that anti-HIV-1 tat antibodies labeled blood vessels from uninfected individuals and that anti-rev antibody stained high endothelial venules. The significance of these findings remains to be examined. In addition to the effects of virus-encoded proteins, the pro- inflammatory molecules released by monocytes must also be considered, both with respect to HIV infection and opportunistic infections such as CMV. Similarly, HIV-injured endothelium may play a role in facilitating entry of opportunistic infections into target tissues. Pulmonary hypertension, with histologic lesions indistinguishable from primary pulmonary hypertension, is a well recognized but rare complication of HIV infection. When present, the pulmonary hypertension contributes significantly to morbidity and mortality. The pathogenesis of this HIV-associated pulmonary hypertension remains unclear, but a direct role of HIV is unlikely, since HIV has not been found in the vascular endothelial cells of these patients. However, an indirect role, secondary to chronic inflammation and immune activation that accompanies HIV infection has been suggested. Speculations include the possibility that HIV infection triggers endothelial cell proliferation in some unknown way, perhaps as a result of stimulation by growth factors. The finding that antiretroviral therapy appears to have a beneficial effect on echocardiographic measures of right heart pressure gradients in HIV- infected patients with pulmonary hypertension is consistent with such an indirect inflammatory etiology. Dysfunction of pulmonary vascular endothelium appears to be important in the pathophysiology of primary pulmonary hypertension and is the probable source of increased platelet activation, release of 5-HT, and thomboxane, as well as reduced the amounts of nitric oxide and prostacyclin. Investigation of the pulmonary vascular endothelium in HIV-associated pulmonary hypertension is likely to clarify the role of endothelial dysfunction in HIV disease, and also lead to a better understanding of primary pulmonary hypertension. Despite the fact that endothelial cells lack CD4 receptors that are thought to be essential for infection with HIV, microvascular endothelial cells from bone marrow have been shown to be capable of infection with the virus. In these cells, IL-1`-induced release of IL-6 and G-CSF is significantly reduced, possibly implicating this deficiency in the hematopoietic malfunction observed during progress of HIV disease. Other reports suggest that umbilical vein endothelial cells may be capable of infection with HIV. These findings illustrate the variable properties of endothelial cells from various tissues and emphasize the need for considering the diversity of endothelial cells in seeking to elucidate the complexities of endothelial-HIV interactions. PROPOSED RESEARCH The emphasis of this solicitation is on how the sequelae of HIV infection alter the expression of endothelial cell genes. The following topics are suggested as examples only. Applicants are urged to consider other topics within the scope of this solicitation based on their own knowledge of the field and their expertise. o Investigation of mechanisms underlying altered gene expression of adhesion molecules in endothelial cells exposed to soluble circulating factors in HIV infection. o Determination of local interactions between tissues and endothelial cells as determinants of endothelial characteristics and susceptibility to injury from HIV. o Elucidation of the signaling mechanisms between monocytes and endothelial cells that enhance HIV replication. o Exploration of the potential for HIV proteins to interact with cell-surface receptors to modify morphologic features and functional activity of endothelial cells. o Investigation of mechanisms underlying the loss of immunocompetence of endothelial cells in HIV infection. o Investigation of the role of the pulmonary vascular endothelium in the pathogenesis of HIV-associated pulmonary hypertension. SPECIAL REQUIREMENTS Upon initiation of the program, the NHLBI will sponsor periodic meetings to encourage exchange of information among investigators who participate in this program. Travel funds for a one day meeting each year, most likely to be held in Bethesda, Maryland should be included in the modules. Applicants should also include a statement in their applications indicating their willingness to participate in these meetings. INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS It is the policy of the NIH that women and members of minority groups and their subpopulations must be included in all NIH supported biomedical and behavioral research projects involving human subjects, unless a clear and compelling rationale and justification is provided that inclusion is inappropriate with respect to the health of the subjects or the purpose of the research. This new policy results from the NIH Revitalization Act of 1993 (Section 492B of Public Law 103-43) and supersedes and strengthens the previous policies (Concerning the Inclusion of Women in Study Populations, and Concerning the Inclusion of Minorities in Study Populations), which have been in effect since 1990. The new policy contains some provisions that are substantially different from the 1990 policies. All investigators proposing research involving human subjects should read the "NIH Guidelines For Inclusion of Women and Minorities as Subjects in Clinical Research," which have been published in the Federal Register of March 20, 1994 (FR 59 14508-14513) and reprinted in the NIH Guide for Grants and Contracts, Volume 23, Number 11, March 18, 1994. Investigators also may obtain copies of the policy from the program staff listed under INQUIRIES. Program staff may also provide additional relevant information concerning the policy. LETTER OF INTENT Prospective applicants are asked to submit, by January 5, 1998, a letter of intent that includes a descriptive title of the proposed research, the name, address, and telephone number of the Principal Investigator, the identities of other key personnel and participating institutions, and the number and title of the RFA in response to which the application may be submitted. Although a letter of intent is not required, is not binding, and does not enter into the review of a subsequent application, the information that it contains allows Institute staff to estimate the potential review workload and avoid conflict of interest in the review. The letter of intent is to be sent to: Dr. C. James Scheirer Chief, Review Branch Division of Extramural Affairs National Heart, Lung, and Blood Institute 6701 Rockledge Dr., Room 7220, MSC 7924 Bethhesda, MD 20892-7924 Telephone: (301) 435-0266 FAX: (301) 480-3541 Email: James_Scheirer@NIH.GOV APPLICATION PROCEDURES Sample budgets and justification page will be provided upon request or following the submission of a letter of intent. BUDGET INSTRUCTIONS The total direct costs must be requested in accordance with the program guidelines and the modifications made to the standard PHS 398 application instructions described below: o DETAILED BUDGET FOR THE INITIAL BUDGET PERIOD Do not complete Form Page 4 of the PHS 398 (rev 5/95). It is not required nor will it be accepted at the time of application. o BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT Do not complete the categorical budget tables on Form page 5 of the PHS 398 (rev. 5/95). Only the requested total direct costs line for each year must be completed based on the number of $25,000 modules being requested. Applicants may not request a change in the amount of each module. A maximum of eight modules ($200,000 direct costs) per year may be requested and each applicant may request up to five years of support for this RFA. Direct cost budgets will remain constant throughout the life of the project (i.e. the same number of modules requested for all budget periods). Any necessary escalation should be considered when determining the number of modules to be requested. However, in the event that the number of modules requested must change in any future year due to the nature of the research proposed, appropriate justification must be provided. Total Direct Costs for the Entire Proposed Project Period should be shown in the box provided. o BUDGET JUSTIFICATION -Budget justifications should be provided under "Justifications" on Form Page 5 of the PHS 398. -List the names, role on the project and proposed percent effort for all project personnel (salaried or unsalaried)and provide a narrative justification for each person based on his/her role on the project. -Identify all consultants by name and organizational affiliation and describe the services to be performed. -Provide a general narrative justification for individual categories (equipment, supplies, etc.) required to complete the work proposed. More detailed justifications should be provided for high cost items. Any large one-time purchases, such as large equipment requests, must be accommodated within these limits. o CONSORTIUM/CONTRACTUAL COSTS - If collaborations or subcontracts are involved that require transfer of funds from the grantee to other institutions, it is necessary to establish formal subcontract agreements with each collaborating institution. A letter of intent from each collaborating institution should be submitted with the application. Only the percentage of the consortium/contractual TOTAL COSTS (direct and indirect) relative to the total DIRECT COSTS of the overall project needs to be stated at this time. The following example should be used to indicate the percentage cost of the consortium, "The consortium agreement represents 27% of overall $175,000 direct costs requested in the first year.". A budget justification for the consortium should be provided as described in the "Budget Justification" section above (no Form Page 5 required for the consortium). Please indicate whether the consortium will be in place for the entire project period and identify any future year changes in the percentage relative to the parent grant. If there is a possibility for an award, the applicant will be requested to identify actual direct and indirect costs for all years of the consortium. Please note that total subcontract costs need not be calculated in $25,000 modules. However, when subcontract funds are added to the parent grant budget, the total direct cost amount must be included in the number of $25,000 modules requested. o BIOGRAPHICAL SKETCH - A biographical sketch is required for all key personnel, following the modified instructions below. Do not exceed the two-page limit for each person. -Complete the educational block at the top of the form page; -List current position(s) and those previous positions directly relevant to the application; -List selected peer-reviewed publications directly relevant to the proposed project, with full citation; -The applicant has the option to provide information on research projects completed and/or research grants participated in during the last five years that are relevant to the proposed project. o OTHER SUPPORT - Do not complete the "Other Support" pages (Form Page 7). Selected other support information relevant to the proposed research may be included in the Biographical Sketch as indicated above. Complete Other Support information will be requested by NHLBI staff if there is a possibility for an award. o CHECKLIST - No "Checklist" page is required as part of the initial application. A completed Checklist will be requested by NHLBI staff if there is a possibility for an award. o The applicant should provide the name and phone number of the individual to contact concerning fiscal and administrative issues if additional information is necessary following the initial review. APPLICATIONS NOT CONFORMING TO THESE GUIDELINES WILL BE CONSIDERED UNRESPONSIVE TO THIS RFA AND WILL BE RETURNED WITHOUT FURTHER REVIEW. Submit a signed, typewritten original of the application and three signed, photocopies, in one package to: DIVISION OF RESEARCH GRANTS NATIONAL INSTITUTES OF HEALTH 6701 ROCKLEDGE DRIVE, ROOM 1040 - MSC 7710 BETHESDA, MD 20892-7710 BETHESDA, MD 20817 (for express courier service) At the time of submission, two additional copies of the application must be sent to Dr. C. James Scheirer, at the same address given above in the section on LETTER OF INTENT. Applications must be received by March 26, 1998. If an application is received after that date, it will be returned to the applicant without review. The Division of Research Grants (DRG) will not accept any application in response to this RFA that is essentially the same as one currently pending initial review, unless the applicant withdraws the pending application. The DRG will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of substantial revisions of applications already re- viewed, but such applications must include an introduction addressing the previous critique. REVIEW CONSIDERATIONS Applications that are complete and responsive to the RFA will be evaluated for scientific and technical merit by an appropriate peer-review group convened in accordance with NIH peer-review procedures. As part of the initial merit review, all applications will receive a written critique and undergo a process in which only those applications deemed to have the highest scientific merit, generally the top half of applications under review, will be discussed, assigned a priority score, and receive a second level review by the National Heart, Lung, and Blood Advisory Council. The personnel category will be reviewed for appropriate staffing based on the requested percent effort. The direct costs budget request will be reviewed for consistency with the proposed methods and specific aims. Any budgetary adjustments recommended by the reviewers will be in $25,000 modules. The duration of support will be reviewed to determine if it is appropriate to ensure successful completion of the requested scope of the project. Other review criteria will include: o scientific, technical or medical significance and originality of proposed research o appropriateness and adequacy of the experimental approach and methodology proposed to carry out the research o qualifications and research experience of the Principal Investigator and staff, particularly, but not exclusively, in the area of the proposed research o availability of the resources necessary to perform the research. The initial review group will also examine the provisions for the protection of human and animal subjects and the safety of the research environment. AWARD CRITERIA Applications will compete for available funds with all other approved applications. The following will be considered in making funding decisions: quality of the proposed project as determined by peer-review, availability of funds, and program priority. INQUIRIES Inquiries concerning this RFA are encouraged. Potential applicants should request a copy of the full RFA which will include sample budget pages as previously stated. The opportunity to clarify any issues or questions from potential applicants is welcome. Direct inquiries regarding programmatic issues to: Dr. Lan-Hsiang Wang Heart Research Program, DHVD National Heart, Lung, and Blood Institute Two Rockledge Center, Suite 9044 6701 Rockledge Drive, MSC 7940 Bethesda, MD 20892-7940 Phone: 301-435-0510 Fax: 301-480-1335 E-mail: LW72F@NIH.GOV Dr. Hannah H. Peavy Division of Lung Diseases National Heart, Lung, and Blood Institute Two Rockledge Center, Suite 10018 6701 Rockledge Drive, MSC 7952 Bethesda, MD 20892-7952 Phone: 301-435-0222 Fax: 301-480-3557 E-mail: hannah_peavy@NIH.GOV Direct inquiries regarding fiscal matters to: Mr. William Darby Grants Operations Branch National Heart, Lung, and Blood Institute Two Rockledge Center, Suite 7128 6701 Rockledge Drive, MSC 7926 Bethesda, MD 20892-7926 Phone: 301-435-0177 Fax: 301-480-3310 E-mail: DARBYW@GWGATE.NHLBI.NIH.GOV AUTHORITY AND REGULATIONS This program is described in the Catalog of Federal Domestic Assistance No. 93.837. Awards are made under authorization of the Public Health Service Act, Title IV, Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC 241 and 285) and administered under PHS grants' policies and Federal Regulations 42 CFR 52 and 45 CFR Part 74. This program is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. The PHS strongly encourages all grant and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of a facility) in which regular or routine education, library, day care, health care or early childhood development services are provided to children. This is consistent with the PHS mission to protect and advance the physical and mental health of the American people. .
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